Nanolipogel comprising a polymeric matrix and a lipid shell

ABSTRACT

Compositions and methods for treating or ameliorating the symptoms of inflammatory or autoimmune disease or disorder are described herein. The compositions contain a nanolipogel for sustained delivery of an effective amount of one or more active agents of choice, preferably a drug for treating or ameliorating the symptoms of inflammatory or autoimmune disease or disorder. The nanolipogel includes a lipid bilayer surrounding a hydrogel core, which may optionally include a host molecule, for example, an absorbent such as a cyclodextrin or ion-exchange resin. In preferred embodiments at least one of active agents is an immunosuppressant.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending application U.S. Ser. No.15/434,971, filed Feb. 16, 2017, which is a continuation of U.S. Ser.No. 14/394,147, filed Oct. 13, 2014, which is a 371 application ofInternational Application No. PCT/US2013/036494, filed Apr. 12, 2013,which claims the benefit of and priority to U.S. Provisional ApplicationNo. 61/623,486, filed Apr. 12, 2012, U.S. Provisional Application No.61/747,624, filed Dec. 31, 2012, and U.S. Provisional Application No.61/747,614, filed Dec. 31, 2012, the disclosures of which are herebyincorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AI056363 awardedby National Institutes of Health and under 0644492 awarded by theNational Science Foundation. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention is generally in the field of compositions andmethods of sustained delivery of high and low molecular weight, orhydrophilic and hydrophobic molecules using core-lipid shell.

BACKGROUND OF THE INVENTION

Autoimmune diseases are broadly characterized by the immunological lossof self-tolerance. In systemic lupus erythematosus (SLE), a canonicalautoimmune disease, a traditional hallmark is the persistence of T and Bcells that are aberrantly reactive to self-antigens such as nucleicacids and nuclear proteins; these T and B lymphocytes promote theproduction of pathogenic autoantibodies which deposit in tissues andprime inflammatory damage (Shlomchik, et al., Nat Rev Immunol, 1(2):147-53 (2001); Rahman, et al., N Engl J Med, 358(9):929-39 (2008)). Thecontribution of innate antigen presenting cells has recently beenelucidated.

Dendritic cells and macrophages have been shown to contribute to lupuspathology by producing proinflammatory cytokines (Blanco, et al.,Science, 294(5546): 1540-3 (2001); Triantafyllopoulou, et al., Proc NatlAcad Sci US A, 107(7):3012-7 (2010)) and promoting expansion ofautoreactive T and B cells (Teichmann, et al., Immunity, 33(6):967-78(2010)).

Current methods used to treat autoimmune diseases have traditionallyrelied on the chronic administration of hydrophobic drugs (Monneaux, etal., Arthritis Res Ther, 11(3):234 (2009)) or, more recently, biologicalagents (proteins and neutralizing antibodies) (Ronnblom, et al., Nat RevRheumatol, 6(6): 339-47 (2010); Navarra, et al., Lancet,377(9767):721-31 (2011); Sfikakis, et al., Curr Opin Rheumatol, 17(5):550-7 (2005)) which inhibit the proliferation or activation oflymphocytes. The conventional administration of pan-immunosuppressivesmall molecule therapies, which are often achieved with hydrophobicdrugs such as cyclophosphamide, azathioprine, or mycophenolate mofetil(MMF), provides therapeutic immunosuppression by blunt reduction oftotal immune cell numbers. This pan-suppressive effect can lead to organtoxicity or lymphopenias and anemia, and render human patients moresusceptible to opportunistic infections (Lee, et al., Lupus,19(6):703-10 (2010); Moroni, et al., Clin J Am Soc Nephrol, 1(5):925-32(2006)). Biological agents which deplete B cells or block T cellcostimulatory signals may provide a more refined, cell-specific approachto immunosuppression, but as a stand-alone monotherapy they may beineffective in attenuating autoimmunity from innate antigen presentingcells.

An ideal therapeutic strategy could combine the pan-suppressive effectsof small molecule therapies with targeting specificity to the immunecells implicated in lupus pathogenesis. Nanoparticles have been activelyexplored for therapeutic use in other diseases such as cancer (Blanco,et al., Cancer Sci, 102(7): 1247-52 (2011)) and infectious pathogens(Look, et al., Adv Drug Deliv Rev, 62(4-5):378-93 (2010)). Thesenanoparticle drug delivery systems can be loaded with therapeuticcompounds with several different methods, and their use in vivo canimprove the bioavailability of therapeutic compounds and to specificallytarget tissues or cells of therapeutic interest (Fahmy, et al.,Materials Today, 8(8): 18-26 (2005)). Few therapeutic strategies haveextensively explored the efficacy of nanoparticles as a drug deliveryvehicle for achieving therapeutic immunosuppression in lupus. To date,published reports regarding nanoparticles and lupus are limited tostudies of nanoparticles that are designed to traffic to relevant sitesof lupus pathology, namely the kidney (Scindia, et al., Arthritis Rheum,58(12):3884-91 (2008); Serkova, et al., Radiology, 255(2):517-26(2010)), but no studies have demonstrated actual therapeutic drugdelivery with these nanoparticle systems. Thus, little is known abouthow nanoparticles may interact with different immune cell sets in lupus,and if these interactions could be exploited to improve lupusimmunotherapies.

Several types of nanoparticle systems, liposomes and synthetic polymericmatrix formulations are commonly used. Several nanoparticle platformsare potential candidates for this purpose. Generally these platforms canbe classified as either vesicular in nature (such as liposomes) orcomposed of solid biodegradable matrices (such as polyester-basednanoparticles). Liposomes are easily modified for encapsulation of smallhydrophilic molecules, and even proteins, but the stability of theseformulations and the release profiles of encapsulated agents can be poor(Maurer, et al., Expert Opinion on Biological Therapy, 1(6):923-947(2001)). Biodegradable solid particles such as those fabricated frompoly(lactic-co-glycolic acid) (PLGA), are highly stable and havecontrollable release characteristics, but pose complications viainduction of maturation of dendritic cells (Yoshida, et al., J BiomedMater Res A, 80(1):7-12 (2007)) and degradation into acidic byproductsthat may promote inflammation (Shive, et al., Adv Drug Deliv Rev,28(1):5-24 (1997)).

No effective treatment for lupus other than generalizedimmunosuppression has been found.

It is therefore an object of the present invention to providecompositions for treating lupus with greater selectivity and efficacy.

It is also an object of the present invention to provide a method fortreatment of lupus with greater selectivity and efficacy.

SUMMARY OF THE INVENTION

Compositions and methods for treating or ameliorating the symptoms ofinflammatory or autoimmune disease or disorder are described herein. Thecompositions contain a nanolipogel for sustained delivery of aneffective amount of one or more active agents of choice, preferably adrug for treating or ameliorating one or more symptoms of aninflammatory or autoimmune disease or disorder. The nanolipogel includesa lipid bilayer surrounding a hydrogel core, which may optionallyinclude a host molecule, for example, an absorbent such as acyclodextrin or ion-exchange resin. In preferred embodiments, at leastone of active agents is an immunosuppressant. In some embodiments, thenanolipogel includes a targeting moiety that increases specificity ofthe particle for activated T cells or antigen presenting cells.

Also provided are methods of incorporating agents into the nanolipogelsdescribed herein. The nanolipogel is loaded with one or more drugs suchthat controlled release of the agent(s) is subsequently achieved. Insome embodiments, the nanolipogel is loaded with one or more firstagent(s) during formation and one or more second agent(s) followingformation by the process of rehydration of the nanolipogel in thepresence of the second agents. The agent(s) can be dispersed within thehydrogel matrix, associated with one or more host molecules, dispersedwithin the liposomal shell, covalently attached to the liposomal shell,and combinations thereof. Drugs can be selectively incorporated at eachof these locales within the nanolipogel.

Also provided herein is a method of treating or ameliorating thesymptoms of an inflammatory or autoimmune disease or disorder using thecompositions described herein. In a preferred embodiment, the treatmentinvolves suppression of both T and B cell effector types. The methodscan include reducing T cell proliferation, activation, response, orfunction, or increasing the tolerance of antigen-presenting cells, orcombinations thereof. For example, the formulations can target andinactivate these immune cells with immunosuppressive drugs at a lowerdose and reduced toxicity compared to conventional drug methods. Thetechnology is generally applicable to a number of inflammatory andautoimmune disease states, for treatment and/or suppression ofautoimmune disease, suppression of allograft rejection and treatment ofallergic diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, comprising FIG. 1A through FIG. 1I, depicts results from exampleexperiments. FIG. 1A and FIG. 1B are schematics of the fabrication ofthe nanolipogel particles (nLG). In FIG. 1A, methacrylate-functionalizedcyclodextrin (CD) was used to solubilize a bioactive such as the TGF-βinhibitor (SB505124). In FIG. 1B, nanolipogels were formulated fromlyophilized liposomes loaded with biodegradable crosslinking polymer,acrylated-drug (CD-SB505) complex, and a second drug such as the peptideIL-2 cytokine. This core-shell structure facilitated entrapment of drugloaded CD and the IL-2 in an interior biodegradable polymer matrix witha PEGylated liposomal exterior. Succinylated β-cyclodextrin (CTD, Inc.)was functionalized with 2-aminoethyl methacrylate (Sigma) by stirring a1:3 molar ratio of the compounds in IX PBS for 1 hour at roomtemperature. The ¹H NMR spectra (500 MHz, D₂O) of SB505124, randomlysuccinylated β-CD, and the inclusion complex of SB505124 with randomlysuccinylated β-CD was determined. The differences observed in thearomatic proton region of SB505124 demonstrate formation of theinclusion complex. The ¹H NMR spectra (500 MHz, D₂O) of rhodamine B,randomly succinylated β-CD, and the inclusion complex of rhodamine Bwith randomly succinylated β-CD showed the differences observed in thearomatic proton region of rhodamine B demonstrate formation of theinclusion complex. FIG. 1C-FIG. 1I show nanolipogel characterization.Nanolipogel size was determined by dynamic light scattering on aZetaPALS instrument (Brookhaven Instruments) in PBS at room temperature.FIG. 1C to FIG. 1E show that encapsulation of SB or SB+IL-2 had nosignificant effect on particle mean diameter or polydispersity. Meandiameter and polydispersity index are representative of 2 lots of eachnanolipogel type (n=10 measurements per sample). The zeta potential ofPC/cholesterol liposomes, PC/cholesterol/PE-PEG-NH₂ liposomes, andnanolipogels were evaluated in 0.1×PBS using a Malvern nanosizer. FIG.1F shows that the zeta potential of liposomes and nanolipogelsincorporating amine-terminated PE-PEG was found to be close to neutral.FIG. 1G shows the composition and formulation properties of thenanolipogel formulation. FIG. 1H shows the polymer structure verified by¹H NMR. Cryo-TEM of nanolipogels demonstrating the formation ofspherical liposomal structures. For TEM analysis, nanolipogel sampleswere stained with osmium tetroxide and then imaged on an FEI TenaiBiotwin microscope. Lipid-specific osmium, tetroxide staining ofcryosectioned samples had a localized staining pattern confined to theexterior membrane of the particle. FIG. 1I shows that thephotopolymerized polymer/CD forms nanoparticulate hydrogel structuresthat are detectable by light scattering even after disruption of theliposomal exterior by detergent.

FIG. 2, comprising FIG. 2A through FIG. 2F, depicts results from exampleexperiments. FIG. 2A-FIG. 2E are comparative release profiles from nLG,lipsomes and solid polymer nanoparticles (PLGA). Cumulative CD- ormethacrylate functionalized-CD (f-CD)-solubilized SB released from nLGsnormalized by initial carrier mass demonstrated that polymerization ofnanolipogels improved the sustained nature of SB release (FIG. 2A).Hydroxypropyl β-CD was used for SB complexation with theunfunctionalized CD. Cumulative IL-2 released determined by ELISA(immunoactive) and by a bioactivity study (bioactive) from nLGsnormalized by initial nanolipogel mass demonstrated that bioactivity ofIL-2 was unaffected by encapsulation (FIG. 2B). Release of SB and IL-2was not affected by incubation of 10 mg nLG in 1 ml full serum (FIG.2C). Comparative cumulative release of SB from liposomes, nanolipogels,and degradable polymeric (poly lactide-co-glycolide) nanoparticles (PLGANPs) demonstrated that incorporation of photo-cured polymer in thenanolipogel vehicle enabled better sustained release and more completerelease of cyclodextrin-solubilized SB (FIG. 2D). PLGA NPs (meandiameter=150+50 nm) were prepared by using a modified water/oil/waterdouble emulsion technique. Liposomes were prepared in an identicalmanner as the nLG without the polymer core. Liposomes were loaded withIL-2 and SB similar to nanolipogels. The diminished percent ofencapsulated SB released from PLGA NPs is attributed to the interactionof the relatively hydrophobic polymer with SB. All particulateformulations were dissolved in 0.1N NaOH+1% SDS to determine 100%release at 7 days (arrow) (FIG. 2D). Comparative cumulative release ofIL-2 from liposomes, nanolipogels, and PLGA NPs demonstrated thatencapsulation of IL-2 in nanolipogels enabled better sustained releaseof cytokine. Cumulative release is presented as % of total IL-2 releasedthrough 7 days. (FIG. 2E) Data in all graphs represent mean oftriplicate samples±1 standard deviation. FIG. 2F compares the sizes andloading of IL-2 and SB in PLGA, nanolipogels and liposomes.

FIG. 3, comprising FIG. 3A through FIG. 3H, depicts results from exampleexperiments. FIG. 3A-FIG. 3H are graphs showing controlled release,clearance, and biodistribution. The distribution of both nanolipogelcarrier and encapsulated drug payload was investigated usingdual-labeled NLG; fluorescein-labeled phosphoethanolamine wasincorporated into the lipid component of rhodamine-loaded nanolipogels.Spectrofluorimetric analysis at 540/625 nm and 490/517 nm showdose-dependent fluorescence with no spectral overlap. FIG. 3A is a graphof cumulative IL-2 (ng/mg nLG) and drug {circumflex over ( )}g SB/mgnLG) released from co-loaded nLGs normalized by carrier mass. Error barsin all plots represent ±1 standard deviation. All experiments wererepeated at least twice with similar results. FIG. 3B is a graph showingclearance (percent of initial dose) of drug dose over time in days:Encapsulation in nanolipogels significantly increased the remainingpercentage of initial dose in the blood at 1 and 24 hours post-injection(two population t test, p<0.01 ###). FIG. 3C and FIG. 3D are graphs ofwhole body distribution. Mice received a single dose of rhodamine-loadednanolipogel or soluble rhodamine (in saline) via intravenous tail veininjection. Animals were sacrificed at 1, 24, 48, and 72 hourspost-injection for extraction and quantification of fluorescence; wholebody biodistribution was determined with rhodamine labeling.Significantly higher (two population t test, p<0.01) amounts ofrhodamine were detected in the major organs of nanolipogel-treatedanimals compared to animals injected with free dye. FIG. 3E is a graphof time dependent accumulation n in subcutaneous tumor: Cumulativerhodamine tumor penetration (circles) after B16 peritumoral injection inB6 mice. Peritumoral tissue was collected to quantify the remaining doseof nLG surrounding the tumor (squares). Controlled release demonstratesrelease of rhodamine, but not lipid (FIG. 3F). Mice bearing subcutaneousB16 tumors received a single IV (tail vein) injection of dual-labeledNLG.

Animals were sacrificed at 1, 2, 3, and 7 days post injection andtissues collected for homogenization, extraction, and quantification ofrhodamine and fluorescein-PE. Analysis of serum showing prolongedcirculation of both encapsulant and delivery vehicle. Similar patternsof biodistribution were observed between lipid (FIG. 3G) and drug payload (FIG. 3H), with highest accumulations of drug occurring in thelungs and liver.

FIG. 4, comprising FIG. 4A through FIG. 4F, depicts results from exampleexperiments. FIG. 4A is a schematic of LED preparation encapsulatingsiRNA/Dendrimer polyplex and drug combinations, with covalentmodification of the outer shell with targeting antibodies or singlechain variable fragments (scFv). FIG. 4B is a graph of the cytotoxicityof LED and LED encapsulating the model drug methotrexate (MTX). Barsindicate successive dilutions of LED or drug or combinations from 1mg/ml to 10 μg/ml. Azide is used as a positive control for cell killing.FIG. 4C is a bar graph showing the % cells exhibiting endosomaldisruption following treatment with unmodified generation 4 PAMAMdendrimers (G4), or dendrimers conjugated to cyclodextrin molecules (CD)that substituted and shielded primary amines with or with FCCP, a smallmolecule ionophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone.FIG. 4D is a bar graph showing the number of GFP positive cells as apercent of total cells transfected with pGFP using various LEDs (G4,G4-3CD, G4-6CD) at various N/P ratios. FIG. 4E is a bar graph showingrelative number of MFICD3+, CD4+ cells control and various LEDsencapsulating different dosages of CD4 or Luciferase siRNA constructs.FIG. 4F is a bar graph showing the level of GFP expression in 293T cellsstably transfected with eGFP following transfection of an siGFPconstruct using LIPOFECTAMINE® or various LEDs containing combinationsof different dendrimer (G)-cyclodextrin conjugates (CDs). This graphmeasures the mean fluorescence intensity (MFI) of GFP to assesssilencing ability of modified dendrimers complexed with siGFP. Thex-axis should read as follows: mock: nonsense siRNA

-   LFA: control siRNA against LFA-   G3: unmodified generation 3 P AMAM dendrimer-   G3 5×: G3 dendrimer with 1 cyclodextrin conjugated (G3-1CD)-   G3 5×d: G3 with 2 CD conjugated (G3-2CD)-   G3 10×: G3 with 3 CD conjugated (G3-3CD)-   G3 20×: G3 with 3.4 CD conjugated (G3-3.4CD)-   G4: G4 dendrimer with no modifications (G4)-   G4 5×: G4 dendrimer with 1 CD conjugated (G4-1CD)-   G4 5×d: G4 dendrimer with 1.3 CD conjugated (G4-1.3CD)-   G4 10×: G4-3CD-   G5: generation 5 (G5) dendrimer with no modifications-   G5 5×: G5-1CD-   G5 10×: G5-3 CD-   G5 10×0.5 mg: G5-3CD, 500 ug used instead of 200 ug in other    treatments G5-   10× D: G5-2.5CD-   G5 20×: G5-4CD

FIG. 5, comprising FIG. 5A through FIG. 5C, depicts results from exampleexperiments. FIG. 5A is a bar graph showing the % MHC-SIINFEKL, murinebone-marrow-derived dendritic cells (BMDCs). MHC-SINFEKL positive cellsfollowing treatment with liposomes containing ovalbumin alone (OVA),dendrimer alone, or a combination of OVA and dendrimer. * p<0.05 byone-way ANOVA Bonferroni post-test. FIG. 5B is a bar graph showing the %MHC-SINFEKL positive cells (by 25.D16-PE staining) following withvarious controls and liposomes includes one or more of dendrimer (i.e.,G5), antigen (i.e., ovalbumin (OVA)), and surface modifications (i.e.,MPLA, and/or CpG) as labeled. The particle formulation containing MPLA,OVA, G5, and CpG was not shown since it encapsulated a prohibitively lowamount of OVA protein, and normalizing treatment groups by the amount ofOVA resulted in cell toxicity because the particle concentration washigher than other groups. FIG. 5C is a bar graph showing the IL-6(pg/mL) expressed from bone marrow dendritic cells (BMDC) treated withLED presenting increasing amounts of CpG.

FIG. 6, comprising FIG. 6A through FIG. 6E, depicts results from exampleexperiments. FIG. 6A is a schematic of paracrine delivery ofimmunosuppressives. The nanolipogels release MPA. The nanoparticles maybe loaded with CTLA4Ig or other biologic, in addition to MPA or otherdrug, and release them. FIG. 6B and FIG. 6C are schematics fornanolipogel particle fabrication for delivery of MPA to cells inpatients with autoimmune disease. FIG. 6C is a depiction of amycophenolic acid (MPA)-cyclodextrin loaded nanolipogel. FIG. 6E is amagnified view of a portion of the nanolipogel of FIG. 6D.

FIG. 7, comprising FIG. 7A through FIG. 7D, depicts results from exampleexperiments. FIG. 7A is a graph showing the particle size distributionof nanolipogels. FIG. 7B is a line graph showing release of MPA fromMPA-loaded nanolipogels over hours. FIG. 7C shows mean diameter (nm),for Nanolipogel in PBS, Nanolipogel in TWEEN 20, liposome in PBS andliposome in TWEEN 20. FIG. 7D shows fluorescence intensity in Jurkatcells treated with free MPA (ng/mL) or the supernatant of PBS containingdrug releasing nanolipogels. The supernatant containednanolipogel-release drug, but no nanolipogel particles. Increase offluorescence intensity correlates with increased proliferation.

FIG. 8, comprising FIG. 8A through FIG. 8F, depicts results from exampleexperiments. FIG. 8A and FIG. 8B are graphs of loading (microgram/mg)(FIG. 8A), percent encapsulation efficiency (FIG. 8B) for liposomeCD-MPA, liposome MPA, nanolipogel CD-MPA, Nanolipogel MPA, and PLPGA MPA(FIG. 8A, FIG. 8B) and Nanolipogel. FIG. 8C and FIG. 8D are graphs ofmedian diameter (nm) of liposomes—empty, loaded, and loaded andcrosslinked FIG. 8C), and nanogel+/−triton X-100, or liposome+/−tritonX-100 (FIG. 8D). FIG. 8E is a bar graph of single particle counting ofnanogel and liposome+/−Triton X-100 treatment. FIG. 8F is a bar graphshowing the percent proliferation for CD4-targeted MPA,isotype-targeted-MPA, non-targeted-MPA, empty particle, MPA and no drug.

FIG. 9, comprising FIG. 9A through FIG. 9E, depicts results from exampleexperiments. FIG. 9A-FIG. 9E are bar graphs of percent body weight (FIG.9A), IU/1 alkaline phosphatase (FIG. 9B), IU/1 alanine transferase (FIG.9C), mg/dl blood urea nitrogen (FIG. 9D), and mg/dl total bilirubin(FIG. 9E), on days 0 (solid bars), 4 (open bars), 7 (grey bars), and 14days (hatched bars) after treatment with buffer, anti-CD4-targetednanolipogels loaded with MPA, non-targeted nanolipogels loaded with MPA,free MPA, non-targeted nanolipogel vehicle, or anti-CD4-targetednanolipogel vehicle.

FIG. 10, comprising FIG. 10A through FIG. 10D, depicts results fromexample experiments. FIG. 10A-FIG. 10D are bar graphs of white bloodcells (K/microliter) (FIG. 10A), platelets (K/microliter) (FIG. 10B),hemoglobin (g/dl) (FIG. 10C), and hematocrit (%) (FIG. 10D) on days 0(solid bars), 4 (open bars), 7 (grey bars), and 14 days (hatched bars)after treatment with buffer, anti-CD4-targeted nanolipogels loaded withMPA, non-targeted nanolipogels loaded with MPA, free MPA, non-targetednanolipogel vehicle, or anti-CD4-targeted nanolipogel vehicle.

FIG. 11, comprising FIG. 11A through FIG. 11C, depicts results fromexample experiments. FIG. 11A and FIG. 11B are two Kaplan-Meier survivalcurves. FIG. 11C is a graph of mean survival age, showing survival overtime (age in weeks) of NZB/W F 1 mice that were treated with alife-long, weekly dose of buffer control (-□-), anti-CD4 nanolipogel-MPA(-▪-), anti-CD4 nanolipogel-vehicle (-⋅-), free MPA (0.625 mpk—same asnanolipogel dose) (-0-), and free MPA (10 mpk—16× nanolipogel dose)(-*-) beginning at 18-20 weeks of age.

FIG. 12, comprising FIG. 12A through FIG. 12I, depicts results fromexample experiments. FIG. 12A-FIG. 12D are graphs of proteinuria (%positive) (FIG. 12A and FIG. 12B) and leukocyte esterase (% positive)(FIG. 12C and FIG. 12D) over time in weeks. FIG. 12A and FIG. 12Ccompare buffer control (-□-), non-targeted Nanolipogel-MPA (-o-), andanti-CD4 nanolipogel-MPA (-•-). FIG. 12B and FIG. 12D compare buffercontrol (-□-), anti-CD4 nanolipogel-MPA (-▪-), anti-CD4nanolipogel-vehicle (-•-), free MPA (0.625 mpk—same as nanolipogel dose)(—⋄—), and free MPA (10 mpk—16× nanolipogel dose) (-▴-). FIG. 12E is abar graph showing elevated blood urea nitrogen (BUN) (as a % elevatedover the physiological reference range of 18-29 mg/dL) followingtreatment with anti-CD4 nanolipogel-MPA, non-targeted Nanolipogel-MPA,buffer, 0.625 mpk free MPA, 10 mpk free MPA, or anti-CD4 Nanolipogelvehicle. FIG. 12F and FIG. 12G are bar graphs showing glomerularnephritis score (FIG. 12F) and interstitial nephritis score (FIG. 12G)for mice treated with anti-CD4 nanolipogel-MPA, non-targetedNanolipogel-MPA, buffer, 0.625 mpk free MPA (same as nanolipogel dose),10 mpk free MPA (16× nanolipogel dose), or anti-CD4 Nanolipogel vehicle.FIG. 12H is a Kaplan-Meier Curve showing percent survival over age forNanolipogel MPA compared to buffer. FIG. 12I is a bar graph showing meansurvival after proteinuria onset (weeks) for Nanolipogel compared tobuffer. Average mouse age of proteinuria onset for nanolipogel MPA of37.2+5.9 weeks compared to buffer of 36.3+5.2 weeks, p<0.05 byMantel-Cox (log-rank) test.

FIG. 13, comprising FIG. 13A through FIG. 13J, depicts results fromexample experiments. FIG. 13A and FIG. 13B are bar graphs of percentrhodamine positive cells within cell subset for spleen (FIG. 13A) andlymph node (FIG. 13B) treated with anti-CD4 nanolipogel (solid);non-targeted Nanolipogel (grey); free rhodamine (open) and PBS(horizontal stripes). FIG. 13C is a bar graph showing the % rhodaminepositive cells within cell subset for ckit+ cells, CD11b+ cells, pDCcells, and cDC cells treated with anti-CD4 nanolipogel (solid);non-targeted Nanolipogel (grey); free rhodamine (open) and PBS(horizontal stripes). FIG. 13D-FIG. 13J are lines graphs of the %initial dose/g organ for anti-CD4 rhodamine Nanolipogel (-•-),non-targeted rhodamine Nanolipogel (-□-), and free rhodamine (-Δ-) forspleen (FIG. 13D), heart (FIG. 13E), lung (FIG. 13F), kidney (FIG. 13G),liver (FIG. 13H), pancreas (FIG. 13I), and serum (FIG. 13J).

FIG. 14, comprising FIG. 14A through FIG. 14E, depicts results fromexample experiments. FIG. 14A-FIG. 14E are graphs of titer (pananti-dsDNA) (FIG. 14A), IgG1 titer (anti-dsDNA) (FIG. 14B), IgG2a(anti-dsDNA) (FIG. 14C), IgG2b Titer (anti-dsDNA) (FIG. 14D), and IgG3titer (anti-dsDNA) (FIG. 14E) over time (age in weeks).

FIG. 15, comprising FIG. 15A through FIG. 15E, depicts results fromexample experiments. FIG. 15A and FIG. 15B are bar graphs of percentageof splenic germinal center B cells (of B22⁺IgD−) (FIG. 15A) and Tfollicular helper cells (defined as CXCR5⁻PD-1⁺ activated CD4 T cells(MI CD26L^(lo), CD44^(hi)) in 36-40-weeks-old NZB/W F 1 mice thatreceived prophylactic therapy with anti-CD4 nanolipogel-MPA,non-targeted Nanolipogel-MPA, buffer, 0.625 mpk free MPA (same asnanolipogel dose), 10 mpk free MPA (16× nanolipogel dose), or anti-CD4Nanolipogel vehicle. The sample size is n=6 to 15 animals per group.Error bars represent the standard error measurement. FIG. 15C-FIG. 15Eare bar graphs the percentage of peripheral blood lymphocytes:plasmablasts (% CD 138^(hi)B220^(neg)) (FIG. 18C), activated CD4 T cells(% CD62L^(low), CD44^(hi) of CD4 T cells) (FIG. 15D), and activated CD 8T cells (% CD62L¹⁰, CD44^(hi) of CD8 T cells) (FIG. 15E), in36-40-weeks-old NZB/W F1 mice that received prophylactic therapy withanti-CD4 nanolipogel-MPA, non-targeted Nanolipogel-MPA, buffer, 0.625mpk free MPA (same as nanolipogel dose), 10 mpk free MPA (16×nanolipogel dose), or anti-CD4 Nanolipogel vehicle beginning at 18-20weeks of age. The sample size is n=6 to 15 animals per group. Error barsrepresent the standard error measurement.

FIG. 16, comprising FIG. 16A through FIG. 16D, depicts results fromexample experiments. FIG. 16A-FIG. 16D are bar graphs of splenocyteswere harvested from 36-40-weeks-old NZB/W F1 mice that received weeklytherapy beginning at 19-20 weeks of age. The graphs show: activated CD4T cells (% CD62L^(low), CD44^(hi) of CD4 T cells) (FIG. 16A), and naiveCD4 T cells (% CD62L^(high), CD44^(low) of CD4 T cells) (FIG. 18D) (FIG.16B), % interferon gamma positive (of CD4 T cells) (FIG. 16C),regulatory T cells (% Foxp3 CD26 (of CD4 T cells) (FIG. 16D), followingtreatment with anti-CD4 nanolipogel-MPA, non-targeted Nanolipogel-MPA,buffer, 0.625 mpk free MPA (same as nanolipogel dose), 10 mpk free MPA(16× nanolipogel dose), or anti-CD4 Nanolipogel vehicle. * p<0.05 bytwo-tailed t-test. d) No differences were observed in CD4 T regulatorycells. The sample size is n=6 to 15 animals per group. Error barsrepresent the standard error measurement.

FIG. 17, comprising FIG. 17A through FIG. 17F, depicts results fromexample experiments. FIG. 17A-FIG. 17F are bar graphs showing the % CD40positive (FIG. 17A, FIG. 17C, and FIG. 17E) or % MHC class II positive(FIG. 17B, FIG. 17D, and FIG. 17F) for conventional DC (CD11C+F4/80−)cells (FIG. 17A and FIG. 17B), macrophages (F4/80+) (FIG. 17C and FIG.17D), or plasmacytoid DC (PDCA-1+F4/80−) cells (FIG. 17E and FIG. 17F),harvested from 36-40-weeks-old mice that received anti-CD4nanolipogel-MPA, non-targeted Nanolipogel-MPA, buffer, 0.625 mpk freeMPA (same as nanolipogel dose), 10 mpk free MPA (16× nanolipogel dose),or anti-CD4 Nanolipogel vehicle beginning at 18-20 weeks of age. Thesample size is n=6 to 15 animals per group. Error bars represent thestandard error measurement.

FIG. 18, comprising FIG. 18A through FIG. 18H, depicts results fromexample experiments. FIG. 18A-FIG. 18F are graphs of bone marrow deriveddendritic cells (BMDCs): % CD40 positive (FIG. 18A), % CD80 positive(FIG. 18B), % CD86 positive (FIG. 18C), interferon gamma (pg/ml) (FIG.18D), IL-12 p70 (pg/ml) (FIG. 18E), TNF-alpha (pg/ml) (FIG. 18F), % MHCclass I positive (FIG. 18G), and % MHC class II positive (FIG. 18H)cultured in vitro for 7 days and treated on day 1 with anti-CD4nanolipogel-MPA, non-targeted Nanolipogel-MPA, anti-CD4 Nanolipogelvehicle, free MPA, and control (blank) all stimulated withlipopolysaccharide (LPS) stimulation (50 ng/mL of LPS for 18 hrbeginning on day 6), or control (blank) no LPS. Results are the averageof 3 separate experiments, with error bars representing the standarderror measurement. * p<0.05 or less by 1-way ANOVA using Bonferronipost-test.

FIG. 19, comprising FIG. 19A through FIG. 19B, depicts results fromexample experiments. FIG. 19A is bar graph showing the level ofinterferon gamma (pg/ml) expressed from CD11c⁺ cells isolated on day 8from the spleens of Balb/c mice injected with treatment on day 0, 3, and7, and subsequently from co-cultured for 4 days with CD4 T cells at aratio of 1×10⁵ dendritic cells to 5×10⁵ T cell. Treatment consisted ofanti-CD4 nanolipogel-MPA, non-targeted Nanolipogel-MPA, anti-CD4Nanolipogel vehicle, free MPA, and control. Error bars represent thestandard deviation, with triplicate measurements from one representativeexperiment shown. * p<0.05 by ANOVA comparison with Bonferronipost-test. This experiment was repeated a total of 3 times, with similartrends. FIG. 19B is a bar graph showing the level of interferon alpha(pg/ml) expressed from bone marrow cells incubated with 1 μg/mL of MP Ain nanogels for 1 hr at 37° C., washed, and then stimulated with CpG-Afor 18 hr. Treatment consisted of anti-CD4 nanolipogel-MPA, non-targetedNanolipogel-MPA, anti-CD4 Nanolipogel vehicle, free MPA, control (blank)with CpG-A and control (blank) without CpG-A. Data are averagedtriplicates in one representative experiment from three repeated trials.Error bars are the standard deviation. * p<0.05 or less by 1-way ANOVAwith Bonferroni multiple comparison post-test.

FIG. 20, comprising FIG. 20A through FIG. 20H, depicts results fromexample experiments. FIG. 20A and FIG. 20B are a Kaplan-Meier survivalcurve (% survival) (FIG. 20A) and a bar graph of mean survival age overtime (age in weeks) (FIG. 20B) of NZB/W F1 mice were treated with buffercontrol (-Δ-), nanolipogel-MPA (-▪-), PLGA-MPA (-•-), and free MPA(-⋄-). FIG. 20C and FIG. 20D are bar graphs showing the % CD40 positive(FIG. 20C) and % CD80 positive (FIG. 20D) out of total dendritic cellstreated with free MPA, or with MPA-loaded or empty nanolipogels (lipo)or PLGA nanoparticles, with no targeting (nt), anti-CD4 targeting, orisotype control targeting following LPS challenge. LPS positive andnegative controls are also included. p<0.05 or less by 1-way ANOVAcomparison. FIGS. 20E, 20F, and 20G are bar graphs showing the IFN-γproduction (pg/ml) (FIG. 20E), IL-12p70 production (FIG. 20F), and TNF-α(pg/ml) (FIG. 20G) by dendritic cells treated with free MPA, or withMPA-loaded or empty nanolipogels (lipo) or PLGA nanoparticles, with notargeting (nt), anti-CD4 targeting, or isotype control targetingfollowing LPS challenge. LPS positive and negative controls are alsoincluded. p<0.05 or less by 1-way ANOVA comparison. FIG. 20H is a linegraph showing dendritic cell internalization (rhodamine positive %) as afunction of nanoparticle dose (pM) for nanolipogels (ngel) or PLGAnanoparticles, with no targeting, anti-CD4 targeting, or isotypecontrol.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“Nanolipogel,” as used herein, refers to a core-shell nanoparticlehaving a polymer matrix core, which can contain a host molecule, withina liposomal shell, which may be unilamellar or bilamellar, optionallycrosslinked.

“Host molecule,” as used herein, refers to a molecule or material whichreversibly associates with an active agent to form a complex. Inparticular embodiments, the host is a molecule that forms an inclusioncomplex with an active agent. Inclusion complexes are formed when anactive agent (i.e., the guest) or portion of an active agent insertsinto a cavity of another molecule, group of molecules, or material(i.e., the host). The host may be a small molecule, an oligomer, apolymer, or combinations thereof. Exemplary hosts includepolysaccharides such as amyloses, cyclodextrins, and other cyclic orhelical compounds containing a plurality of aldose rings, for example,compounds formed through 1,4 and 1,6 bonding of monosaccharides (such asglucose, fructose, and galactose) and disaccharides (such as sucrose,maltose, and lactose). Other exemplary host compounds include cryptands,cryptophanes, cavitands, crown ethers, dendrimers, ion-exchange resins,calixarenes, valinomycins, nigericins, catenanes, polycatenanes,carcerands, cucurbiturils, and spherands.

“Small molecule,” as used herein, refers to molecules with a molecularweight of less than about 2000 g/mol, more preferably less than about1500 g/mol, most preferably less than about 1200 g/mol.

“Hydrogel,” as used herein, refers to a water-swellable polymeric matrixformed from a three-dimensional network of macromolecules held togetherby covalent or non-covalent crosslinks, that can absorb a substantialamount of water (by weight) to form a gel.

“Hydrodynamic radius” of a particle, as used herein, is the radius of ahard and perfectly spherical object of the same mass and having the samerate of diffusion as the particle. This may also be referred to,interchangeably, as the Stokes radius or as the Stokes-Einstein radius.Diameter is typically two times the radius.

“Nanoparticle”, as used herein, generally refers to a particle having adiameter from about 10 nm up to, but not including, about 1 micron,preferably from 100 nm to about 1 micron. The particles can have anyshape. Nanoparticles having a spherical shape are generally referred toas “nanospheres”.

“Molecular weight” as used herein, generally refers to the relativeaverage chain length of the bulk polymer, unless otherwise specified. Inpractice, molecular weight can be estimated or characterized usingvarious methods including gel permeation chromatography (GPC) orcapillary viscometry. GPC molecular weights are reported as theweight-average molecular weight (Mw) as opposed to the number-averagemolecular weight (Mn). Capillary viscometry provides estimates ofmolecular weight as the inherent viscosity determined from a dilutepolymer solution using a particular set of concentration, temperature,and solvent conditions.

“Mean particle size” as used herein, generally refers to the statisticalmean particle size (diameter) of the particles in a population ofparticles. The diameter of an essentially spherical particle may referto the physical or hydrodynamic diameter. The diameter of anon-spherical particle may refer preferentially to the hydrodynamicdiameter. As used herein, the diameter of a non-spherical particle mayrefer to the largest linear distance between two points on the surfaceof the particle. Mean particle size can be measured using methods knownin the art, such as dynamic light scattering.

“Monodisperse” and “homogeneous size distribution”, are usedinterchangeably herein and describe a population of nanoparticles ormicroparticles where all of the particles are the same or nearly thesame size. As used herein, a monodisperse distribution refers toparticle distributions in which 90% of the distribution lies within 15%of the median particle size, more preferably within 10% of the medianparticle size, most preferably within 5% of the median particle size.

“Active Agent”, as used herein, refers to a physiologically orpharmacologically active substance that acts locally and/or systemicallyin the body. An active agent is a substance that is administered to apatient for the treatment (e.g., therapeutic agent), prevention (e.g.,prophylactic agent), or diagnosis (e.g., diagnostic agent) of a diseaseor disorder.

The term “immune cell” refers to cells of the innate and acquired immunesystem including neutrophils, eosinophils, basophils, monocytes,macrophages, dendritic cells, lymphocytes including B cells, T cells,and natural killer cells.

II. Nanolipogels

Nanolipogels are core-shell nanoparticulates that combine the advantagesof both liposomes and polymer-based particles for sustained delivery ofactive agents. As discussed in more detail below, typically, the outershell protects cargo, provides biocompatibility and a surface forfunctionalization with targeting molecule(s). The outer shellencapsulates components such that they are not exposed until desired,for example, in response to environmental conditions or stimuli,creatings monodisperse, reproducible particle populations, and mediatinginternalization into desired cell types. The inner core, which can be adendrimer or other polymer, has separate and additive functionalities toouter shell. For example, the inner shell allows for secondarydeposition of drug, vaccine, or imaging agent; increases loading ofcomponents with different physiochemical properties into the particle;allows for tunable release of contents from particles; increasescytosolic availability of DNA/RNA, drug, and/or protein by disruptingendosomes, all leading to enhanced drug effects, antigen presentation,and transfection/silencing.

Nanolipogels have a polymer matrix core containing one or more hostmolecules. The polymeric matrix is preferably a hydrogel, such as acrosslinked block copolymer containing one or more poly(alkylene oxide)segments, such as polyethylene glycol, and one or more aliphaticpolyester segments, such as polylactic acid. One or more host molecules,such as a cyclodextrin, dendrimer, or ion exchange resin, is dispersedwithin or covalently bound to the polymeric matrix. The hydrogel core issurrounded by a liposomal shell.

Nanolipogels can be constructed to incorporate a variety of activeagents that can subsequently be released in a controlled fashion. Activeagents can be dispersed within the hydrogel matrix, associated with oneor more host molecules, dispersed within the liposomal shell, covalentlyattached to the liposomal shell, and combinations thereof. Active agentscan be selectively incorporated at each of these locales within thenanolipogel. Furthermore, the release rate of active agents from each ofthese locales can be independently tuned. Because each of these localespossesses distinct properties, including size andhydrophobicity/hydrophilicity, the chemical entities independentlyincorporated at each of these locales can differ dramatically withrespect to size and composition. For example, nanolipogels can be loadedwith one or more proteins dispersed within the polymeric matrix as wellas small molecule hydrophobic drugs associated with host molecules.

In this way, nanolipogels can provide simultaneous sustained release ofagents that differ widely in chemical composition and molecular weight.In a non-limiting example, nanolipogels may be loaded with both ahydrophobic, small molecule antigen associated with a host molecule andan immunoadjuvant, such as an immunostimulatory protein, dispersedwithin the polymeric matrix. These nanolipogels can provide sustainedrelease of the antigen together with the adjuvant, to optimize an immuneresponse. In a particular example, simultaneous sustained delivery bynanolipogels of an immunostimulatory protein, Interleukin-2 (IL-2), aswell as a low molecular weight organic molecule,2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride, an inhibitor of transforming growth factor-β (TGF-β), wasachieved. This construct leads to an antitumor response in a murinesystem that is far superior to that achievable with the administrationin solution of either agent alone or a combination of the two.Additionally, nanolipogels can favorably modulate biodistribution of oneor more active agents encapsulated therein.

Nanolipogels are typically spherical in shape, with average particlesizes ranging between about 50 nm and about 1000 nm, more preferablybetween about 75 nm and about 300 nm, most preferably between about 90nm and about 200 nm. In certain embodiments, the nanolipogels possess anaverage particle size between about 100 nm and about 140 nm. Particlesmay be non-spherical. Depending upon the nature of the lipids present inthe liposomal shell of the nanolipogels, the nanolipogels having apositive, negative, or near neutral surface charge may be prepared. Incertain embodiments, the nanolipogels possess a near neutral surfacecharge. In certain embodiments, the nanolipogels possess a ζ-potentialof between about 10 mV and about −10 mV, more preferably between about 5mV and about −5 mV, more preferably between about 3 mV and about −3 mV,most preferably between about 2 mV and about −2 mV.

A. Core

The nanolipogel core is formed from a polymeric matrix and one or morehost molecules. The nanolipogel core may further include one or moreactive agents. The active agents may be complexed to the host molecules,dispersed with polymeric matrix, or combinations thereof.

1. Polymeric Matrix

The polymeric matrix of the nanolipogels may be formed from one or morepolymers or copolymers. By varying the composition and morphology of thepolymeric matrix, one can achieve a variety of controlled releasecharacteristics, permitting the delivery of moderate constant doses ofone or more active agents over prolonged periods of time.

The polymeric matrix may be formed from non-biodegradable orbiodegradable polymers; however, preferably, the polymeric matrix isbiodegradable. The polymeric matrix can be selected to degrade over atime period from ranging from one day to one year, more preferably fromseven days to 26 weeks, more preferably from seven days to 20 weeks,most preferably from seven days to 16 weeks.

In general, synthetic polymers are preferred, although natural polymersmay be used. Representative polymers include poly(lactic acid),poly(glycolic acid), poly(lactic acid-co-glycolic acids),polyhydroxyalkanoates such as poly3-hydroxybutyrate orpoly4-hydroxybutyrate; polycaprolactones; poly(orthoesters);polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones);poly(glycolide-co-caprolactones); polycarbonates such as tyrosinepolycarbonates; polyamides (including synthetic and natural polyamides),polypeptides, and poly(amino acids); polyesteramides; otherbiocompatible polyesters; poly(dioxanones); poly(alkylene alkylates);hydrophilic polyethers; polyurethanes; polyetheresters; polyacetals;polycyanoacrylates; polysiloxanes; poly(oxyethylene)/poly(oxypropylene)copolymers; polyketals; polyphosphates; polyhydroxyvalerates;polyalkylene oxalates; polyalkylene succinates; poly(maleic acids),polyvinyl alcohols, polyvinylpyrrolidone; poly(alkylene oxides) such aspolyethylene glycol (PEG); derivativized celluloses such as alkylcelluloses (e.g., methyl cellulose), hydroxyalkyl celluloses (e.g.,hydroxypropyl cellulose), cellulose ethers, cellulose esters,nitrocelluloses, polymers of acrylic acid, methacrylic acid orcopolymers or derivatives thereof including esters, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate) (jointly referred to herein as“polyacrylic acids”), as well as derivatives, copolymers, and blendsthereof.

As used herein, “derivatives” include polymers having substitutions,additions of chemical groups and other modifications to the polymericbackbones described above routinely made by those skilled in the art.Natural polymers, including proteins such as albumin, collagen, gelatin,prolamines, such as zein, and polysaccharides such as alginate andpectin, may also be incorporated into the polymeric matrix. While avariety of polymers may be used to form the polymeric matrix, generally,the resulting polymeric matrix will be a hydrogel. In certain cases,when the polymeric matrix contains a natural polymer, the naturalpolymer is a biopolymer which degrades by hydrolysis, such as apolyhydroxyalkanoate.

In preferred embodiments, the polymeric matrix contains one or morecrosslinkable polymers. Preferably, the crosslinkable polymers containone or more photo-polymerizable groups, allowing for the crosslinking ofthe polymeric matrix following nanolipogel formation. Examples ofsuitable photo-polymerizable groups include vinyl groups, acrylategroups, methacrylate groups, and acrylamide groups. Photo-polymerizablegroups, when present, may be incorporated within the backbone of thecrosslinkable polymers, within one or more of the sidechains of thecrosslinkable polymers, at one or more of the ends of the crosslinkablepolymers, or combinations thereof.

The polymeric matrix may be formed from polymers having a variety ofmolecular weights, so as to form nanolipogels having properties,including drug release rates, optimal for specific applications.Generally, the polymers which make up the polymeric matrix possessaverage molecular weights of about 500 Da and 50 kDa. In cases where thepolymeric matrix is formed from non-crosslinkable polymers, the polymerstypically possess average molecular weights ranging between about 1 kDaand about 50 kDa, more preferably between about 1 kDa and about 70 kDa,most preferably between about 5 kDa and about 50 kDa. In cases where thepolymeric matrix is formed from crosslinkable polymers, the polymerstypically possess lower average molecular weights ranging between about500 Da and about 25 kDa, more preferably between about 1 kDa and about10 kDa, most preferably between about 3 kDa and about 6 kDa. Inparticular embodiments the polymeric matrix is formed from acrosslinkable polymer having an average molecular weight of about 5 kDa.

In some embodiments, the polymeric matrix is formed from a poly(alkyleneoxide) polymer or a block copolymer containing one or more poly(alkyleneoxide) segments. The poly(alkylene oxide) polymer or poly(alkyleneoxide) polymer segments may contain between 8 and 500 repeat units, morepreferably between 40 and 300 repeat units, most preferably between 50and 150 repeat units. Suitable poly(alkylene oxides) includepolyethylene glycol (also referred to as polyethylene oxide or PEG),polypropylene 1,2-glycol, poly(propylene oxide), polypropylene1,3-glycol, and copolymers thereof.

In some embodiments, the polymeric matrix is formed from an aliphaticpolyester or a block copolymer containing one or more aliphaticpolyester segments. Preferably the polyester or polyester segments arepoly(lactic acid) (PLA), poly(glycolic acid) PGA, orpoly(lactide-co-glycolide) (PLGA). In preferred embodiments, thepolymeric matrix is formed from a block copolymer containing one or morepoly(alkylene oxide) segments, one or more aliphatic polyester segments,and optionally one or more photo-polymerizable groups. In these cases,the one or more poly(alkylene oxide) segments imbue the polymer with thenecessary hydrophilicity, such that the resultant polymer matrix forms asuitable hydrogel, while the polyester segments provide a polymericmatrix with tunable hydrophobicity/hydrophilicity and/or the desired invivo degradation characteristics.

The degradation rate of the polyester segments, and often thecorresponding drug release rate, can be varied from days (in the case ofpure PGA) to months (in the case of pure PLA), and may be readilymanipulated by varying the ratio of PLA to PGA in the polyestersegments. In addition, the poly(alkylene oxides), such as PEG, andaliphatic polyesters, such as PGA, PLA, and PLGA have been establishedas safe for use in humans; these materials have been used in humanclinical applications, including drug delivery system, for more than 30years.

In certain embodiments, the polymeric matrix is formed from a tri-blockcopolymer containing a central poly(alkylene oxide) segment, adjoiningaliphatic polyester segments attached to either end of the centralpoly(alkylene oxide) segment, and one or more photo-polymerizablegroups. Preferably, the central poly(alkylene oxide) segment is PEG, andaliphatic polyesters segments are PGA, PLA, or PLGA.

Generally, the average molecular weight of the central poly(alkyleneoxide) segment is greater than the average molecular weight of theadjoining polyester segments. In certain embodiments, the averagemolecular weight of the central poly(alkylene oxide) segment is at leastthree times greater than the average molecular weight of one of theadjoining polyester segments, more preferably at least five timesgreater than the average molecular weight of one of the adjoiningpolyester segments, most preferably at least ten times greater than theaverage molecular weight of one of the adjoining polyester segments.

In some cases, the central poly(alkylene oxide) segment possesses anaverage molecular weight ranging between about 500 Da and about 10,000Da, more preferably between about 1,000 Da and about 7,000 Da, mostpreferably between about 2,500 Da and about 5,000 Da. In particularembodiments, average molecular weight of the central poly(alkyleneoxide) segment is about 4,000 Da. Typically, each adjoining polyestersegment possesses an average molecular weight ranging between about 100Da and about 5,000 Da, more preferably between about 100 Da and about1,000 Da, most preferably between about 100 Da and about 500 Da.

In a preferred embodiment, the polymeric matrix is formed from thetri-block copolymer shown below

where m and n are, independently for each occurrence, integers between 1and 500, more preferably between 10 and 150.

Examples of preferred natural polymers include proteins such as albumin,collagen, gelatin and prolamines, for example, zein, and polysaccharidessuch as alginate, cellulose derivatives and polyhydroxyalkanoates, forexample, polyhydroxybutyrate. The in vivo stability of themicroparticles can be adjusted during the production by using polymerssuch as poly(lactide-co-glycolide) copolymerized with polyethyleneglycol (PEG). If PEG is exposed on the external surface, it may increasethe time these materials circulate due to the hydrophilicity of PEG.

Examples of preferred non-biodegradable polymers include ethylene vinylacetate, poly(meth)acrylic acid, polyamides, copolymers and mixturesthereof.

The matrix can also be made of gel-type polymers, such as alginate,produced through traditional ionic gelation techniques. The polymers arefirst dissolved in an aqueous solution, mixed with barium sulfate orsome bioactive agent, and then extruded through a microdroplet formingdevice, which in some instances employs a flow of nitrogen gas to breakoff the droplet. A slowly stirred (approximately 100-170 RPM) ionichardening bath is positioned below the extruding device to catch theforming microdroplets. The microparticles are left to incubate in thebath for twenty to thirty minutes in order to allow sufficient time forgelation to occur.

Microparticle particle size is controlled by using various sizeextruders or varying either the nitrogen gas or polymer solution flowrates. Chitosan microparticles can be prepared by dissolving the polymerin acidic solution and crosslinking it with tripolyphosphate.Carboxymethyl cellulose (CMC) microparticles can be prepared bydissolving the polymer in acid solution and precipitating themicroparticle with lead ions. In the case of negatively charged polymers(e.g., alginate, CMC), positively charged ligands (e.g., polylysine,polyethyleneimine) of different molecular weights can be ionicallyattached.

Perhaps the most widely used are the aliphatic polyesters, specificallythe hydrophobic poly (lactic acid) (PLA), more hydrophilic poly(glycolic acid) PGA and their copolymers, poly (lactide-co-glycolide)(PLGA). The degradation rate of these polymers, and often thecorresponding drug release rate, can vary from days (PGA) to months(PLA) and is easily manipulated by varying the ratio of PLA to PGA.Second, the physiologic compatibility of PLGA and its hompolymers PGAand PLA have been established for safe use in humans; these materialshave a history of over 30 years in various human clinical applicationsincluding drug delivery systems. PLGA nanoparticles can be formulated ina variety of ways that improve drug pharmacokinetics and biodistributionto target tissue by either passive or active targeting. Themicroparticles are designed to release molecules to be encapsulated orattached over a period of days to weeks. Factors that affect theduration of release include pH of the surrounding medium (higher rate ofrelease at pH 5 and below due to acid catalyzed hydrolysis of PLGA) andpolymer composition. Aliphatic polyesters differ in hydrophobicity andthat in turn affects the degradation rate. Specifically the hydrophobicpoly (lactic acid) (PLA), more hydrophilic poly (glycolic acid) PGA andtheir copolymers, poly (lactide-co-glycolide) (PLGA) have variousrelease rates. The degradation rate of these polymers, and often thecorresponding drug release rate, can vary from days (PGA) to months(PLA) and is easily manipulated by varying the ratio of PLA to PGA.

2. Host Molecules

Host molecules are molecules or materials which reversibly associatewith an active agent to form a complex. By virtue of their ability toreversibly form complexes with active agents, host molecules canfunction to control the release of a complexed active agent in vivo.

In some cases, the host molecule is a molecule that forms an inclusioncomplex with an active agent. Inclusion complexes are formed when anactive agent (i.e., the guest), or portion of an active agent, insertsinto a cavity of another molecule, group of molecules, or material(i.e., the host). Typically, the guest molecule associates with the hostmolecule without affecting the framework or structure of the host. Forexample, in the case of inclusion complexes, the size and shape of theavailable cavity in the host molecule remain substantially unaltered asa consequence of complex formation. The host molecule may be a smallmolecule, an oligomer, a polymer, or combinations thereof. Exemplaryhosts include polysaccharides such as amyloses, cyclodextrins, and othercyclic or helical compounds containing a plurality of aldose rings, forexample, compounds formed through 1,4 and 1,6 bonding of monosaccharides(such as glucose, fructose, and galactose) and disaccharides (such assucrose, maltose, and lactose). Other exemplary host compounds includecryptands, cryptophanes, cavitands, crown ethers, dendrimers,ion-exchange resins, calixarenes, valinomycins, nigericins, catenanes,polycatenanes, carcerands, cucurbiturils, and spherands.

In still other embodiments, organic host compounds or materials includecarbon nanotubes, fullerenes, and/or graphene-based host materials.Carbon nanotubes (CNTs) are allotropes of carbon with a cylindricalnanostructure. Nanotubes are members of the fullerene structural family,which also includes the spherical buckyballs, and the ends of a nanotubemay be capped with a hemisphere of the buckyball structure. Their nameis derived from their long, hollow structure with the walls formed byone-atom-thick sheets of carbon, called graphene. These sheets arerolled at specific and discrete (“chiral”) angles, and the combinationof the rolling angle and radius decides the nanotube properties.Nanotubes can be categorized as single-walled nanotubes (SWNTs) andmulti-walled nanotubes (MWNTs). Nanotubes and/or fullerenes can serve ashosts, for example, by encapsulating or entrapping the material to bedelivered (i.e., the guest) within the tubes or fullerenes.Alternatively, the exterior and/or interior of the tubes and/orfullerenes can be functionalized with functional groups which cancomplex to the guest to be delivered. Complexations include, but are notlimited to, ionic interactions, hydrogen bonding, Van der Waalsinteractions, and pi-pi interactions, such as pi-stacking.

Graphenes are also an allotrope of carbon. The structure of graphene isa one-atom-thick planar sheet of sp²-bonded carbon atoms that aredensely packed in a honeycomb crystal lattice. Graphene is the basicstructural element of some carbon allotropes including graphite,charcoal, carbon nanotubes and fullerenes. The guest to be delivered canassociate with and/or complex to graphene or functionalized graphene asdescribed above for nanotubes and fullerenes.

The host material can also be an inorganic material, including but notlimited to, inorganic phosphates and silica.

Suitable host molecules are generally selected for incorporation intonanolipogels in view of the identity of the active agent(s) to bedelivered and the desired drug release profile. In order to form acomplex with the active agent being delivered, the host molecule isgenerally selected to be complimentary to the active agent both in termsof sterics (size) and electronics (charge and polarity). For example, inthe case of host molecules that form inclusion complexes with the activeagent to be delivered, the host molecule will typically possess anappropriately-sized cavity to incorporate the active agent. In addition,the host molecule typically possesses a cavity of appropriatehydrophobicity/hydrophilicity to promote complex formation with theactive agent. The strength of the guest-host interaction will influencethe drug release profile of the active agent from the nanolipogel, withstronger guest-host interactions generally producing more prolonged drugrelease.

Generally, the host molecules are dispersed within the polymeric matrixthat forms the nanolipogel core. In some cases, one or more hostmolecules are covalently coupled to the polymeric matrix. For example,the host molecules may be functionalized with one or more pendantreactive functional groups that react with the polymer matrix. Inparticular embodiments, the host molecules contain one or more pendantreactive functional groups that react with the polymer matrix tocrosslink the polymer matrix. Examples of suitable reactive functionalgroups include methacrylates, acrylates, vinyl groups, epoxides,thiiranes, azides, and alkynes.

In certain embodiments, the host molecule is a cyclodextrin.Cyclodextrins are cyclic oligosaccharides containing six(α-cyclodextrin), seven (β-cyclodextrin), eight (γ-cyclodextrin), ormore α-(1,4)-linked glucose residues. The hydroxyl groups of thecyclodextrins are oriented to the outside of the ring while theglucosidic oxygen and two rings of the non-exchangeable hydrogen atomsare directed towards the interior of the cavity. As a result,cyclodextrins possess a hydrophobic inner cavity combined with ahydrophilic exterior. Upon combination with a hydrophobic active agent,the active agent (i.e., the guest) inserts into the hydrophobic interiorof the cyclodextrin (i.e., the host).

The cyclodextrin may be chemically modified such that some or all of theprimary or secondary hydroxyl groups of the macrocycle, or both, arefunctionalized with one or more pendant groups. The pendant groups maybe reactive functional groups that can react with the polymeric matrix,such as methacrylates, acrylates, vinyl groups, epoxides, thiiranes,azides, alkynes, and combinations thereof. The pendant groups may alsoserve to modify the solubility of the cyclodextrin. Exemplary groups ofthis type include sulfinyl, sulfonyl, phosphate, acyl, and C₁-C₁₂ alkylgroups optionally substituted with one or more (e.g., 1, 2, 3, or 4)hydroxy, carboxy, carbonyl, acyl, oxy, and oxo groups. Methods ofmodifying these alcohol residues are known in the art, and manycyclodextrin derivatives are commercially available.

Examples of suitable cyclodextrins include α-cyclodextrin;β-cyclodextrin; γ-cyclodextrin; methyl α-cyclodextrin; methylβ-cyclodextrin; methyl γ-cyclodextrin; ethyl β-cyclodextrin; butylα-cyclodextrin; butyl β-cyclodextrin; butyl γ-cyclodextrin; pentylγ-cyclodextrin; hydroxy ethyl β-cyclodextrin; hydroxyethylγ-cyclodextrin; 2-hydroxypropyl α-cyclodextrin; 2-hydroxypropylβ-cyclodextrin; 2-hydroxypropyl γ-cyclodextrin; 2-hydroxybutylβ-cyclodextrin; acetyl α-cyclodextrin; acetyl β-cyclodextrin; acetylγ-cyclodextrin; propionyl β-cyclodextrin; butyryl β-cyclodextrin;succinyl α-cyclodextrin; succinyl β-cyclodextrin; succinylγ-cyclodextrin; benzoyl β-cyclodextrin; palmityl β-cyclodextrin;toluenesulfonyl β-cyclodextrin; acetyl methyl β-cyclodextrin; acetylbutyl β-cyclodextrin; glucosyl α-cyclodextrin; glucosyl β-cyclodextrin;glucosyl γ-cyclodextrin; maltosyl α-cyclodextrin; maltosylβ-cyclodextrin; maltosyl γ-cyclodextrin; α-cyclodextrincarboxymethylether; β-cyclodextrin carboxymethylether; γ-cyclodextrincarboxymethylether; carboxymethylethyl β-cyclodextrin; phosphate esterα-cyclodextrin; phosphate ester β-cyclodextrin; phosphate esterγ-cyclodextrin; 3-trimethylammonium-2-hydroxypropyl β-cyclodextrin;sulfobutyl ether β-cyclodextrin; carboxymethyl α-cyclodextrin;carboxymethyl β-cyclodextrin; carboxymethyl γ-cyclodextrin, andcombinations thereof.

Preferred cyclodextrins include α-cyclodextrins, β-cyclodextrins, andγ-cyclodextrins functionalized with one or more pendant acrylate ormethacrylate groups. In a particular embodiment, the host molecule is aβ-cyclodextrin functionalized with multiple methacrylate groups. Anexemplary host molecule of this type is illustrated below, wherein Rrepresents a C₁-C₆ alkyl group.

As a further example, the host molecule may also be a material thattemporarily associates with an active agent via ionic interactions. Forexample, conventional ion exchange resins known in the art for use incontrolled drug release may serve as host molecules. See, for example,Chen, et al. “Evaluation of ion-exchange microspheres as carriers forthe anticancer drug doxorubicin: in vitro studies.” J. Pharm. Pharmacol.44(3):211-215 (1992) and Farag, et al. “Rate of release of organiccarboxylic acids from ion exchange resins” J. Pharm. Sci. 77(10):872-875(1988).

By way of exemplification, when the active agent being delivered is acationic species, suitable ion exchange resins may include a sulfonicacid group (or modified sulfonic acid group) or an optionally modifiedcarboxylic acid group on a physiologically acceptable scaffold.Similarly, where the active agent is an anionic species, suitable ionexchange resins may include amine-based groups (e.g., trimethylamine fora strong interaction, or dimethylethanolamine for a weaker interaction).Cationic polymers, such as polyethyleneimine (PEI), can function as hostmolecules for complex oligonucleotides such as siRNA. In other cases,the host molecule is a dendrimer, such as a poly(amidoamine) (PAMAM)dendrimer. Cationic and anionic dendrimers can function as hostmaterials by ionically associating with active agents, as describedabove. In addition, medium-sized dendrimers, such as three- andfour-generation PAMAM dendrimers, may possess internal voids spaceswhich can accommodate active agents, for example, by complexation ofnucleic acids.

In some embodiments the host molecule is a dendrimer conjugated to acyclodextrin. In some embodiments, the cyclodextrin(s) shields primaryamines of dendrimer. Suitable dendrimers and cyclodextrins are discussedabove. Unmodified dendrimer (i.e., generation 4 PAMAM dendrimer (G4))was empirically better at endosomal disruption than dendrimer conjugatedwith cyclodexrin (CD) (See the Examples below). Without being bound bytheory, it is believed that terminal amine groups on PAMAM dendrimersprovide endosomal buffering and disrupt endosomes by the proton spongeeffect. Accordingly, increasing CD results in a decrease in endosomaldisruption. As discussed in the Examples below, different combinationsof dendrimers and cyclodextrins can be used to modulate the transfectionefficiency and level of endosomal disruption in the cell.

Preferably, the one or more host molecules are present in an amount offrom about 0.1% to about 40% w/w of the polymeric matrix, morepreferably from about 0.1% to about 25% w/w of the overall formulation.

3. Active Agents

Active agents to be delivered include therapeutic, nutritional,diagnostic, and prophylactic agents. The active agents can be smallmolecule active agents or biomacromolecules, such as proteins,polypeptides, or nucleic acids. Suitable small molecule active agentsinclude organic and organometallic compounds, as well as steroids,chemotherapeutic or cytoxic compounds, radioisotype or radioactivematerial, macrolides, both naturally occurring and synthetic analogs,derivative, or other forms of these compounds. . . . The small moleculeactive agents can be a hydrophilic, hydrophobic, or amphiphiliccompound.

Exemplary therapeutic agents that can be incorporated into nanolipogelsinclude tumor antigens, CD4+ T-cell epitopes, cytokines,chemotherapeutic agents, radionuclides, small molecule signaltransduction inhibitors, photothermal antennas, monoclonal antibodies,immunologic danger signaling molecules, other immunotherapeutics,enzymes, antibiotics, antivirals (especially protease inhibitors aloneor in combination with nucleosides for treatment of HIV or Hepatitis Bor C), anti-parasites (helminths, protozoans), growth factors, growthinhibitors, hormones, hormone antagonists, antibodies and bioactivefragments thereof (including humanized, single chain, and chimericantibodies), antigen and vaccine formulations (including adjuvants),peptide drugs, anti-inflammatories, immunomodulators (including ligandsthat bind to Toll-Like Receptors to activate the innate immune system,molecules that mobilize and optimize the adaptive immune system,molecules that activate or up-regulate the action of cytotoxic Tlymphocytes, natural killer cells and helper T cells, and molecules thatdeactivate or down-regulate suppressor or regulatory T cells), agentsthat promote uptake of nanolipogels into cells (including dendriticcells and other antigen-presenting cells), nutraceuticals such asvitamins, and oligonucleotide drugs (including DNA, RNAs, antisense,aptamers, small interfering RNAs, ribozymes, external guide sequencesfor ribonuclease P, and triplex forming agents).

Exemplary diagnostic agents include paramagnetic molecules, fluorescentcompounds, magnetic molecules, and radionuclides, x-ray imaging agents,and contrast agents.

In certain embodiments, the nanolipogel includes one or more anticanceragents. Representative anti-cancer agents include, but are not limitedto, alkylating agents (such as cisplatin, carboplatin, oxaliplatin,mechlorethamine, cyclophosphamide, chlorambucil, dacarbazine, lomustine,carmustine, procarbazine, chlorambucil and ifosfamide), antimetabolites(such as fluorouracil (5-FU), gemcitabine, methotrexate, cytosinearabinoside, fludarabine, and floxuridine), antimitotics (includingtaxanes such as paclitaxel and decetaxel and vinca alkaloids such asvincristine, vinblastine, vinorelbine, and vindesine), anthracyclines(including doxorubicin, daunorubicin, valrubicin, idarubicin, andepirubicin, as well as actinomycins such as actinomycin D), cytotoxicantibiotics (including mitomycin, plicamycin, and bleomycin),topoisomerase inhibitors (including camptothecins such as camptothecin,irinotecan, and topotecan as well as derivatives of epipodophyllotoxinssuch as amsacrine, etoposide, etoposide phosphate, and teniposide),antibodies to vascular endothelial growth factor (VEGF) such asbevacizumab (AVASTIN®), other anti-VEGF compounds; thalidomide(THALOMID®) and derivatives thereof such as lenalidomide (REVLIMID®);endostatin; angiostatin; receptor tyrosine kinase (RTK) inhibitors suchas sunitinib (SUTENT®); tyrosine kinase inhibitors such as sorafenib(Nexavar®), erlotinib (Tarceva®), pazopanib, axitinib, and lapatinib;transforming growth factor-α or transforming growth factor-β inhibitors,and antibodies to the epidermal growth factor receptor such aspanitumumab (VECTIBIX®) and cetuximab (ERBITUX®).

In certain embodiments, the nanolipogel includes one or moreimmunomodulatory agents. Exemplary immunomodulatory agents includecytokines, xanthines, interleukins, interferons, oligodeoxynucleotides,glucans, growth factors (e.g., TNF, CSF, GM-CSF and G-CSF), hormonessuch as estrogens (diethylstilbestrol, estradiol), androgens(testosterone, HALOTESTIN® (fluoxymesterone)), progestins (MEGACE®(megestrol acetate), PROVERA® (medroxyprogesterone acetate)), andcorticosteroids (prednisone, dexamethasone, hydrocortisone).

In one embodiment, the active agent is a therapeutic used to treatautoimmune diseases such as rheumatoid arthritis and lupus.

Nonsteroidal anti-inflammatory drugs (NSAIDs), which are administered tohelp ease symptoms like pain, swelling and stiffness, can be used. Themost common used NSAIDs are ibuprofen and naproxen.

Disease-modifying anti-rheumatic drugs (DMARDs), are agents which slowdown—or even halt—the progress of a disease. The workhorse of this groupis methotrexate. Other DMARDs include sulfasalazine (brand nameAzulfidine) and leflunomide (Arava).

The major category within biologies is tumor necrosis factor (TNF)blockers, which counteract high levels of inflammatory proteins.Etanercept (Enbrel), infliximab (Remicade) and adalimumab (Humira) arethe most widely used. Another promising group is interleukin-1 (IL-1)blockers like anakinra (Kineret).

Corticosteroids include prednisolone, hydrocortisone,methylprednisolone, dexamethasone, cortisone, triamcinolone, andbetamethasone.

Mycophenolate mofetil (MMF) and its active metabolite mycophenolic acid(MPA) are both very effective immunosuppressive agent. MMF has been usedto treat autoimmune and inflammatory skin diseases. Lipsky, Lancet,348:L1357-1359 (1996) and has become a valuable therapeutic option inchildren with autoimmune disease. Filler, et al., Pediatric Rheumatol,8: 1 (2010). Mycophenolic acid (MP A) is a relatively new adjuvant drugthat selectively inhibits T and B lymphocyte proliferation bysuppressing de novo purine synthesis. Other steroid sparingimmunosuppressive agents include azathioprine, methotrexate andcyclophosphamide. In a preferred embodiment, the activeimmunosuppressant is MPA. The examples show hydrogel-based nanoparticlesthat were loaded with the immunosuppressant drug mycophenolic acid(MPA).

MPA is the active form of mycophenolate mofetil, which is currently usedas an immunosuppressant in humans for lupus and other autoimmune diseasetherapy (Ginzler, et al., NEngl J Med, 353(21):2219-28 (2005)). We choseto encapsulate MPA in nanoparticles because it has broadimmunosuppressive effects on several immune cell types. MPA blocks thede novo synthesis pathway of guanine nucleotides. T and B cellproliferation is acutely impaired by MPA because these cells lack thebiosynthetic salvage pathways that could circumvent impaired de novoguanine production (Jonsson, et al., Clin Exp Immunol, 124(3): 486-91(2001); Quemeneur, et al., J Immunol, 169(5):2747-55 (2002); Jonsson, etal., Int Immunopharmacol, 3(1):31-7 (2003); and Karnell, et al., JImmunol, 187(7): 3603-12 (2011). Furthermore, MPA can impair theactivation of dendritic cells and their ability to stimulate alloantigenresponses (Mehling, et al., J Immunol, 165(5):2374-81 (2000); Lagaraine,et al., Int Immunol, 17(4):351-63 (2005); and Wadia, et al., HumImmunol, 70(9):692-700 (2009)), and has been suggested to promote thedevelopment of tolerogenic dendritic cells (Lagaraine, et al., JLeukocBiol, 84(4): 1057-64 (2008)). Like many immunosuppressant drugs, MPA isvery hydrophobic, with a reported partition coefficient (log P value) of3.88 (Elbarbry, et al., J Chromatogr B Analyt Technol Biomed Life Sci,859(2): 276-81 (2007)).

In a preferred embodiment demonstrated by the examples, the hostmolecule is used to deliver a hydrophobic agent for example, theimmunosuppressant, MPA. An immunosuppressant may include any smallmolecule that suppresses the function of the immune system or thatincreases susceptibility to infectious diseases. In certain preferredembodiments, the immunosuppressant is an inhibitor of T cellproliferation, an inhibitor of B cell proliferation, or an inhibitor ofT cell and B cell proliferation. In certain embodiments the T cell or Bcell proliferation inhibitors inhibit or regulate the synthesis ofguanine monophosphate. For example, the immunosuppressant can bemycophenolic acid, the structure of which is shown below.

Alternatively, the immunosuppressant is a prodrug of mycophenolic acidincluding, but not limited to, mycophenolate mofetil (marketed under thetrade names CELLCEPT® by the Swedish company F. Hoffmann-La Roche Ltd.),the structure of which is shown below.

A salt of the immunosuppressant may also be used, for example, a salt ofmycophenolic acid includes, but is not limited to, the mycophenolatesodium (marketed under the trade name MYFORTIC® by Novartis). In certainpreferred embodiments the immunosuppressant is a synthetically modifiedanalogue of mycophenolic acid.

In some embodiments, the immunosuppressant is a purine analogueincluding, but not limited to, azathioprine (marketed under a variety oftrade names including AZASAN® by Salix and IMURAN® by GlaxoSmithKline)or mercaptopurine (marketed under the trade name PURTNETHOL®((Mercaptopurine). In some embodiments the immunosuppressant is anantimetabolite that inhibits the use and/or the synthesis of purines,such as a purine nucleoside phosphorylase inhibitor.

Intravenous immunoglobulin (IVIg), which is a blood product made up ofantibodies that is delivered by IV, is used to get the immune systemback on track without suppressing its normal function.

Examples of immunological adjuvants that can be associated with theparticles include, but are not limited to, TLR ligands, C-Type LectinReceptor ligands, NOD-Like Receptor ligands, RLR ligands, and RAGEligands. TLR ligands can include lipopolysaccharide (LPS) andderivatives thereof, as well as lipid A and derivatives there ofincluding, but not limited to, monophosphoryl lipid A (MPL),glycopyranosyl lipid A, PET-lipid A, and 3-O-desacyl-4′-monophosphoryllipid A. In a specific embodiment, the immunological adjuvant is MPL. Inanother embodiment, the immunological adjuvant is LPS. TLR ligands canalso include, but are not limited to, TLR3 ligands (e.g.,polyinosinic-polycytidylic acid (poly(I:C)), TLR7 ligands (e.g.,imiquimod and resiquimod), and TLR9 ligands.

The nanolipogel may also include antigens and/or adjuvants (i.e.,molecules enhancing an immune response). Peptide, protein, and DNA basedvaccines may be used to induce immunity to various diseases orconditions. Cell-mediated immunity is needed to detect and destroyvirus-infected cells. Most traditional vaccines (e.g. protein-basedvaccines) can only induce humoral immunity. DNA-based vaccine representsa unique means to vaccinate against a virus or parasite because a DNAbased vaccine can induce both humoral and cell-mediated immunity. Inaddition, DNA based vaccines are potentially safer than traditionalvaccines. DNA vaccines are relatively more stable and morecost-effective for manufacturing and storage. DNA vaccines consist oftwo major components—DNA carriers (or delivery vehicles) and DNAsencoding antigens. DNA carriers protect DNA from degradation, and canfacilitate DNA entry to specific tissues or cells and expression at anefficient level.

In certain embodiments, the nanolipogel core contains two or more activeagents. In preferred embodiments, the nanolipogel core contains both asmall molecule hydrophobic active agent, preferably associated with oneor more suitable host molecules, and a hydrophilic active agentdispersed within the polymeric matrix. In particular embodiments, thehydrophilic active agent is a protein, such as a therapeutic cytokine.By incorporating a hydrophobic active agent in association with a hostmolecule and a hydrophilic molecule dispersed within the polymericmatrix, controlled release of two or more active agents, including twoor more active agents with varied physiochemical characteristics (suchas solubility, hydrophobicity/hydrophilicity, molecular weight, andcombinations thereof) can be achieved.

In a preferred embodiment demonstrated by the examples, the hostmolecule is used to deliver a low molecular weight compounds such as anchemotherapeutic, where the host molecule retards release of the lowmolecular weight compound, and a larger hydrophilic compound, such as acytokine, so that release of both molecules occurs over a similar timeperiod.

B. Shell Components

Nanolipogels include a liposomal shell composed of one or moreconcentric lipid monolayers or lipid bilayers. The shell can furtherinclude one or active agents, targeting molecules, or combinationsthereof.

1. Lipids

Nanolipogels include a liposomal shell composed of one or moreconcentric lipid monolayers or lipid bilayers. The composition of theliposomal shell may be varied to influence the release rate of one ormore active agents in vivo. The lipids may also be covalentlycrosslinked, if desired, to alter in vivo drug release.

The lipid shell can be formed from a single lipid bilayer (i.e., theshell may be unilamellar) or several concentric lipid bilayers (i.e.,the shell may be multilamellar). The lipid shell may be formed from asingle lipid; however, in preferred embodiments, the lipid shell isformed from a combination of more than one lipid. The lipids can beneutral, anionic or cationic lipids at physiologic pH.

Suitable neutral and anionic lipids include sterols and lipids such ascholesterol, phospholipids, lysolipids, lysophospholipids, andsphingolipids. Neutral and anionic lipids include, but are not limitedto, phosphatidylcholine (PC) (such as egg PC, soy PC), including1,2-diacyl-glycero-3-phosphocholines; phosphatidylserine (PS),phosphatidylglycerol, phosphatidylinositol (PI); glycolipids;sphingophospholipids, such as sphingomyelin; sphingoglycolipids (alsoknown as 1-ceramidyl glucosides), such as ceramide galactopyranoside,gangliosides and cerebrosides; fatty acids, sterols containing acarboxylic acid group such as cholesterol or derivatives thereof; and1,2-diacyl-sn-glycero-3-phosphoethanolamines, including1,2-dioleoyl-sn-Glycero-3-phosphoethanolamine or 1,2-dioleolylglycerylphosphatidylethanolamine (DOPE), 1,2-dihexadecylphosphoethanolamine(DHPE), 1,2-distearoylphosphatidylcholine (DSPC),1,2-dipalmitoylphosphatidylcholine (DPPC), and1,2-dimyristoylphosphatidylcholine (DMPC). Also suitable are natural(e.g., tissue derived L-α-phosphatidyl: egg yolk, heart, brain, liver,soybean) and/or synthetic (e.g., saturated and unsaturated1,2-diacyl-sw-glycero-3-phosphocholines,1-acyl-2-acyl-sn-glycero-3-phosphocholines,1,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of theselipids. Suitable cationic lipids includeN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, alsoreferred to as TAP lipids, for example as a methylsulfate salt. SuitableTAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP(dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Othersuitable cationic lipids include dimethyldioctadecyl ammonium bromide(DDAB), 1,2-diacyloxy-3-trimethylammonium propanes,N-[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine (DODAP),1,2-diacyloxy-3-dimethylammonium propanes,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),1,2-dialkyloxy-3-dimethylammonium propanes,dioctadecylamidoglycylspermine (DOGS),3-[N—(N′,N′-dimethylamino-ethane)carbamoyl]cholesterol (DC-Choi);2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanaminiumtrifluoro-acetate (DOSPA), 3-alanyl cholesterol, cetyltrimethylammoniumbromide (CTAB), diC₁₄-amidine,N-tert-butyl-N′-tetradecyl-3-tetradecylamino-propionamidine,N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG),ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride,1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), andN,N,N′,N′-tetramethyl-,N′-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butanediammonium iodide,142-(acyloxy)ethylp-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazoliniumchloride derivatives, such as1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazoliniumchloride (DOTIM) and1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazoliniumchloride (DPTIM), and 2,3-dialkyloxypropyl quaternary ammoniumderivatives containing a hydroxyalkyl moiety on the quaternary amine,for example, 1,2-dioleoyl-3-dimethyl-hydroxy ethyl ammonium bromide(DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DOME), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide(DOME-HP), 1,2-dioleyl-oxy-propyl-3-dimethyl-hydroxybutyl ammoniumbromide (DOME-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentylammonium bromide (DORIE-Hpe),1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide(DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammoniumbromide (DPRIE), and 1,2-disteryloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DSRIE).

Other suitable lipids include PEGylated derivatives of the neutral,anionic, and cationic lipids described above. Incorporation of one ormore PEGylated lipid derivatives into the lipid shell can result in ananolipogel which displays polyethylene glycol chains on its surface.The resulting nanolipogels may possess increased stability andcirculation time in vivo as compared to nanolipogels lacking PEG chainson their surfaces. Examples of suitable PEGylated lipids includedistearoylphosphatidylethanlamine-polyethylene glycol (DSPE-PEG),including DSPE PEG (2000 MW) and DSPE PEG (5000 MW),dipalmitoyl-glycero-succinate polyethylene glycol (DPGS-PEG),stearyl-polyethylene glycol and cholesteryl-polyethylene glycol.

In preferred embodiments, the lipid shell is formed from a combinationof more than one lipid. In certain embodiments the lipid shell is formedfrom a mixture of at least three lipids. In particular embodiments, thelipid shell is formed from a mixture of phosphatidyl choline (PC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG), and cholesterol.

In some embodiments, the lipid shell is formed from a mixture of one ormore PEGylated phospholipids and one or more additional lipids orsterols. In certain instances, the molar ratio of the one or morePEGylated lipids to the one or more additional lipids or sterols rangesfrom about 1:1 to about 1:6, more preferably from about 1:2 to about1:6, most preferably from about 1:3 to about 1:5. In particularembodiments, the molar ratio of the one or more PEGylated lipids to theone or more additional lipids or sterols is about 1:4. In someembodiments, the lipid shell is formed from a mixture of one or morephospholipids and one or more additional lipids or sterols. In certaininstances, the molar ratio of the one or more phospholipids to the oneor more additional lipids or sterols ranges from about 1:1 to about 6:1,more preferably from about 2:1 to about 6:1, most preferably from about3:1 to about 5:1. In particular embodiments, the molar ratio of the oneor more phospho lipids to the one or more additional lipids or sterolsis about 4:1.

In a preferred embodiments, the lipid shell is formed from a mixture ofa phospholipid, such as phosphatidyl choline (PC), a PEGylatedphospholipid, such as1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG), and cholesterol. In particular embodiments,the lipid shell is formed from a mixture of phosphatidyl choline,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG), and cholesterol in a 3:1:1 molar ratio.

2. Targeting Molecules and Molecules Decreasing RES Uptake

The surface of the nanolipogels, or the core host, can be modified tofacilitate targeting through the attachment of targeting molecules.

Exemplary target molecules include proteins, peptides, nucleic acids,lipids, saccharides, or polysaccharides that bind to one or more targetsassociated with an organ, tissue, cell, or extracellular matrix, orspecific type of tumor or infected cell. The degree of specificity withwhich the nanolipogels are targeted can be modulated through theselection of a targeting molecule with the appropriate affinity andspecificity. For example, a targeting moiety can be a polypeptide, suchas an antibody that specifically recognizes a tumor marker that ispresent exclusively or in higher amounts on a malignant cell (e.g., atumor antigen). Suitable targeting molecules that can be used to directnanoparticles to cells and tissues of interest, as well as methods ofconjugating target molecules to nanoparticles, are known in the art.See, for example, Ruoslahti, et al. Nat. Rev. Cancer, 2:83-90 (2002).Targeting molecules can also include neuropilins and endothelialtargeting molecules, integrins, selectins, and adhesion molecules.Targeting molecules can be covalently bound to nanolipogels using avariety of methods known in the art.

In certain embodiments, the liposomal shell includes one or morePEGylated lipids. The PEG, or other hydrophilic polyalkylene oxide,avoids uptake of the lipogels by the reticuloendothelial system (“RES”),thereby prolonging in vivo residence time.

The surface of the nanolipogels can be modified to facilitate targetingthrough the attachment of targeting molecules. These can be proteins,peptides, nucleic acid molecules, saccharides or polysaccharides thatbind to a receptor or other molecule on the surface of a targeted cell.The degree of specificity can be modulated through the selection of thetargeting molecule. For example, antibodies are very specific. These canbe polyclonal, monoclonal, fragments, recombinant, or single chain, manyof which are commercially available or readily obtained using standardtechniques. T-cell specific molecules and antigens which are bound byantigen presenting cells as well as tumor targeting molecules can bebound to the surface of the nanolipogel and/or to the host molecule. Thetargeting molecules may be conjugated to the terminus of one or more PEGchains present on the surface of the liposomal shell.

3. Active Agents

The shell of the nanolipogels may optionally contain one or more activeagents, including any of the active agents described above.

Hydrophobic active agents, such as proteins, may be covalently connectedto the surface of the nanolipogel, whereas hydrophilic active agents maybe covalently connected to the surface of the nanolipogel or dispersedwithin the liposomal shell. In certain embodiments, the liposomal shellincludes one or more PEGylated lipids. In these cases, one or moreactive agents may be conjugated to the terminus of one or more PEGchains present on the surface of the liposomal shell. In particularembodiments, one or more active agents are covalently connected to thesurface of the nanolipogel via a linking group that is cleaved inresponse to an external chemical or physical stimulus, such as a changein ambient pH, so as to trigger release of the active agent at a desiredphysiological locale.

III. Methods of Manufacture, Loading, and Pharmaceutical Compositions

A. Methods of Manufacture and Loading

“Nanolipogel”, a nanoparticle that combines the advantages of bothliposomes and polymer-based particles for sustained delivery of nucleicacids, proteins and small molecules. The nanolipogel can be in the formof spheres, discs, rods or other geometries with different aspectratios. The nanosphere can be larger, i.e., microparticles. Thenanolipogel is typically formed of synthetic or natural polymers capableof encapsulating agents by remote loading and tunable in properties soas to facilitate different rates of release. Release rates are modulatedby varying the polymer to lipid ratio from 0.05 to 5.0, more preferablyfrom 0.5 to 1.5.

Nanolipogels are designed to be loaded with agents either prior to,during or after formation and subsequently function ascontrolled-release vehicles for the agents. The nanolipogel can beloaded with more than one agent such that controlled release of themultiplicity of agents is subsequently achieved.

The nanolipogel is loaded with one or more first agents during formationand one or more second agents following formation by the process ofrehydration of the nanolipogel in the presence of the second agents. Forexample, the nanolipogel is loaded with a molecule that serves as anadjuvant and the nanolipogel thereafter incorporates one or more targetantigens after formation, for the controlled release of adjuvanttogether with the antigens. Alternatively, the nanolipogel loaded withadjuvant is inserted into the site of a tumor in a patient, the tumor isablated, the nanolipogel is loaded with released tumor antigens and thenanolipogel releases the tumor antigens together with adjuvant into thebody of the patient in a controlled manner.

In another embodiment, the nanolipogel is loaded with an antigen, amolecule that serves as an adjuvant and a targeting molecule for antigenpresenting cells, the nanolipogel is taken up by antigen presentingcells and the antigen is appropriately processed and presented toT-helper and cytotoxic T-cells to promote a cell-mediated immuneresponse.

In yet another embodiment, the nanolipogel loaded with a molecule thatserves as an adjuvant and a targeting molecule for antigen presentingcells is inserted into the site of a tumor in a patient, the tumor isablated and the nanolipogel is loaded with released tumor antigens, thenanolipogel is taken up by antigen presenting cells and the releasedtumor antigens are appropriately processed and presented to T-helper andcytotoxic T-cells to promote a cell-mediated immune response.

B. Pharmaceutical Compositions

Pharmaceutical compositions including nanolipogels are provided.Pharmaceutical compositions can be for administration by parenteral(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), transdermal (either passively or using iontophoresis orelectroporation), or transmucosal (nasal, vaginal, rectal, orsublingual) routes of administration or using bioerodible inserts andcan be formulated in dosage forms appropriate for each route ofadministration.

In some embodiments, the compositions are administered systemically, forexample, by intravenous or intraperitoneal administration, in an amounteffective for delivery of the compositions to targeted cells. Otherpossible routes include trans-dermal or oral.

In certain embodiments, the compositions are administered locally, forexample, by injection directly into a site to be treated. In someembodiments, the compositions are injected or otherwise administereddirectly to one or more tumors. Typically, local injection causes anincreased localized concentration of the compositions which is greaterthan that which can be achieved by systemic administration. In someembodiments, the compositions are delivered locally to the appropriatecells by using a catheter or syringe. Other means of delivering suchcompositions locally to cells include using infusion pumps (for example,from Alza Corporation, Palo Alto, Calif.) or incorporating thecompositions into polymeric implants (see, for example, P. Johnson andJ. G. Lloyd-Jones, eds., Drug Delivery Systems (Chichester, England:Ellis Horwood Ltd., 1987), which can effect a sustained release of thenanolipogels to the immediate area of the implant.

The nanolipogels can be provided to the cell either directly, such as bycontacting it with the cell, or indirectly, such as through the actionof any biological process. For example, the nanolipogels can beformulated in a physiologically acceptable carrier or vehicle, andinjected into a tissue or fluid surrounding the cell. The nanolipogelscan cross the cell membrane by simple diffusion, endocytosis, or by anyactive or passive transport mechanism. As further studies are conducted,information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age, and generalhealth of the recipient, will be able to ascertain proper dosing. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment desired.Generally dosage levels of 0.001 to 10 mg/kg of body weight daily areadministered to mammals. Generally, for intravenous injection orinfusion, dosage may be lower. Generally, the total amount of thenanolipogel-associated active agent administered to an individual willbe less than the amount of the unassociated active agent that must beadministered for the same desired or intended effect.

1. Formulations for Parenteral Administration

In a preferred embodiment the nanolipogels are administered in anaqueous solution, by parenteral injection.

The formulation can be in the form of a suspension or emulsion. Ingeneral, pharmaceutical compositions are provided including effectiveamounts of one or more active agents optionally include pharmaceuticallyacceptable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers. Such compositions can include diluents sterile water,buffered saline of various buffer content (e.g., Tris-HCl, acetate,phosphate), pH and ionic strength; and optionally, additives such asdetergents and solubilizing agents (e.g., TWEEN® 20, TWEEN® 80 alsoreferred to as polysorbate 20 or 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzylalcohol) and bulking substances (e.g., lactose, mannitol). Examples ofnon-aqueous solvents or vehicles are propylene glycol, polyethyleneglycol, vegetable oils, such as olive oil and corn oil, gelatin, andinjectable organic esters such as ethyl oleate. The formulations may belyophilized and redissolved/resuspended immediately before use. Theformulation may be sterilized by, for example, filtration through abacteria retaining filter, by incorporating sterilizing agents into thecompositions, by irradiating the compositions, or by heating thecompositions.

2. Formulations for Topical and Mucosal Administration

The nanolipogels can be applied topically. Topical administration caninclude application to the lungs, nasal, oral (sublingual, buccal),vaginal, or rectal mucosa. These methods of administration can be madeeffective by formulating the shell with transdermal or mucosal transportelements. For transdermal delivery such elements may include chemicalenhancers or physical enhancers such as electroporation or microneedledelivery. For mucosal delivery PEGylation of the outer shell or additionof chitosan or other mucosal permeants or PH protective elements fororal delivery.

Compositions can be delivered to the lungs while inhaling and traverseacross the lung epithelial lining to the blood stream when deliveredeither as an aerosol or spray dried particles having an aerodynamicdiameter of less than about 5 microns.

A wide range of mechanical devices designed for pulmonary delivery oftherapeutic products can be used, including but not limited to,nebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices are the Ultravent® nebulizer(Mallinckrodt Inc., St. Louis, Mo.); the Acorn® II nebulizer (MarquestMedical Products, Englewood, Colo.); the Ventolin® metered dose inhaler(Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler® powderinhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkindall have inhalable insulin powder preparations approved or in clinicaltrials where the technology could be applied to the formulationsdescribed herein.

Formulations for administration to the mucosa will typically be spraydried drug particles, which may be incorporated into a tablet, gel,capsule, suspension or emulsion. Standard pharmaceutical excipients areavailable from any formulator. Oral formulations may be in the form ofchewing gum, gel strips, tablets, capsules, or lozenges. Oralformulations may include excipients or other modifications to theparticle which can confer enteric protection or enhanced deliverythrough the GI tract, including the intestinal epithelia and mucosa (seeSamstein, et al., Biomaterials, 29(6):703-8 (2008). Transdermalformulations may also be prepared. These will typically be ointments,lotions, sprays, or patches, all of which can be prepared using standardtechnology. Transdermal formulations can include penetration enhancers.Chemical enhancers and physical methods including electroporation andmicroneedles can work in conjunction with this method.

IV. Methods of Treatment

The methods of treatment disclosed herein typically include using ananolipogel loaded with one or more active agents, to deliver the one ormore active agents into a cell, or to a cell's microenvironment. Themethods typically include contacting the active agent-loaded nanolipogelwith one more cells. The contacting can occur in vivo or in vitro.

Administration of a drug or other cargo to cells or a subject usingnanolipogels can be compared to a control, for example, delivery of thedrug or other cargo to cells or a subject using conventional deliverymethods such as free cargo/drug delivery, delivery using conventionalPLGA nanoparticles, or delivery using conventional liposomal methodssuch as LIPOFECTAMINE®. Nanolipogels can be used to deliver cargo to atarget cells with increased efficacy compared to conventional deliverymethods. In some embodiments less cargo or drug is required whendelivered using nanolipogels compared to conventional delivery methodsto achieve the same or greater therapeutic benefit.

In some embodiments toxicity is reduced or absent compared toconventional delivery methods. For example, in some embodiments, whiteblood cell, platelet, hemoglobin, and hematocrit levels were withinnormal physiological ranges; no liver or renal toxicities are observed;body weight and serum concentrations for alkaline phosphatase, alaninetransferase, total bilirubin, and blood urea nitrogen are normal; orcombinations thereof following administration of loaded nanolipogels tothe subject.

A. In Vivo Methods

The disclosed compositions can be used in a method of delivering activeagents to cells in vivo. In some in vivo approaches, the compositionsare administered to a subject in a therapeutically effective amount. Asused herein the term “effective amount” or “therapeutically effectiveamount” means a dosage sufficient to treat, inhibit, or alleviate one ormore symptoms of the disorder being treated or to otherwise provide adesired pharmacologic and/or physiologic effect. The precise dosage willvary according to a variety of factors such as subject-dependentvariables (e.g., age, immune system health, etc.), the disease, and thetreatment being effected.

1. Drug Delivery

The particles described herein can be used to deliver an effectiveamount of one or more therapeutic, diagnostic, and/or prophylacticagents to a patient in need of such treatment. The amount of agent to beadministered can be readily determine by the prescribing physician andis dependent on the age and weight of the patient and the disease ordisorder to be treated.

The particles are useful in drug delivery (as used herein “drug”includes therapeutic, nutritional, diagnostic and prophylactic agents),whether injected intravenously, subcutaneously, or intramuscularly,intraperitoneally, administered to the nasal or pulmonary system,injected into a site of inflammation, administered to a mucosal surface(vaginal, rectal, buccal, sublingual), or encapsulated for oraldelivery. The particles may be administered as a dry powder, as anaqueous suspension (in water, saline, buffered saline, etc), in ahydrogel, organogel, in capsules, tablets, troches, or other standardpharmaceutical excipients. The preferred embodiments are aqueoussuspensions and dry powders that have been resuspended into aqueous/gelform. As discussed herein, compositions can be used to as deliveryvehicles for a number of active agents including small molecules,nucleic acids, proteins, and other bioactive agents. The active agent oragents can be encapsulated within, dispersed within, and/or associatedwith the surface of the nanolipogel particle. In some embodiments, thenanolipogel packages two, three, four, or more different active agentsfor simultaneous delivery to a cell.

2. Transfection

The disclosed compositions can be for cell transfection ofpolynucleotides. As discussed in more detail below, the transfection canoccur in vitro or in vivo, and can be applied in applications includinggene therapy and disease treatment. The compositions can be moreefficient, less toxic, or a combination thereof when compared to acontrol. In some embodiments, the control is cells treated with analternative transfection reagent such as LIPOFECTAMINE 2000.

The particular polynucleotide delivered by the nanolipogel can beselected by one of skill in the art depending on the condition ordisease to be treated. The polynucleotide can be, for example, a gene orcDNA of interest, a functional nucleic acid such as an inhibitory RNA, atRNA, an rRNA, or an expression vector encoding a gene or cDNA ofinterest, a functional nucleic acid a tRNA, or an rRNA. In someembodiments two or more polynucleotides are administered in combination.In some embodiments, the polynucleotide encodes a protein.

In some embodiments, the polynucleotide is not integrated into the hostcell's genome (i.e., remains extrachromosomal). Such embodiments can beuseful for transient or regulated expression of the polynucleotide, andreduce the risk of insertional mutagenesis. Therefore, in someembodiments, the nanolipogels are used to deliver mRNA ornon-integrating expression vectors that are expressed transiently in thehost cell.

In some embodiments, the polynucleotide is integrated into the hostcell's genome. For example, gene therapy is a technique for correctingdefective genes responsible for disease development. Researchers may useone of several approaches for correcting faulty genes: (a) a normal genecan be inserted into a nonspecific location within the genome to replacea nonfunctional gene. This approach is most common; (b) an abnormal genecan be swapped for a normal gene through homologous recombination; (c)an abnormal gene can be repaired through selective reverse mutation,which returns the gene to its normal function; (d) the regulation (thedegree to which a gene is turned on or off) of a particular gene can bealtered.

Gene therapy can include the use of viral vectors, for example,adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, AIDS virus, neuronal trophic virus, Sindbis and other RNAviruses, including these viruses with the HIV backbone. Also useful areany viral families which share the properties of these viruses whichmake them suitable for use as vectors. Typically, viral vectors contain,nonstructural early genes, structural late genes, an RNA polymerase IIItranscript, inverted terminal repeats necessary for replication andencapsidation, and promoters to control the transcription andreplication of the viral genome. When engineered as vectors, virusestypically have one or more of the early genes removed and a gene orgene/promoter cassette is inserted into the viral genome in place of theremoved viral DNA.

Gene targeting via target recombination, such as homologousrecombination (HR), is another strategy for gene correction. Genecorrection at a target locus can be mediated by donor DNA fragmentshomologous to the target gene (Hu, et al., Mol. Biotech., 29: 197-210(2005); Olsen, et al., J. Gene Med., 7: 1534-1544 (2005)). One method oftargeted recombination includes the use of triplex-formingoligonucleotides (TFOs) which bind as third strands tohomopurine/homopyrimidine sites in duplex DNA in a sequence-specificmanner. Triplex forming oligonucleotides can interact with eitherdouble-stranded or single-stranded nucleic acids.

Methods for targeted gene therapy using triplex-forming oligonucleotides(TFO's) and peptide nucleic acids (PNAs) are described in U.S. PublishedApplication No. 20070219122 and their use for treating infectiousdiseases such as HIV are described in U.S. Published Application No.2008050920. The triplex-forming molecules can also be tail clamp peptidenucleic acids (tcPNAs), such as those described in U.S. PublishedApplication No. 2011/0262406. Highly stable PNA:DNA:PNA triplexstructures can be formed from strand invasion of a duplex DNA with twoPNA strands. In this complex, the PNA/DNA/PNA triple helix portion andthe PNA/DNA duplex portion both produce displacement of thepyrimidine-rich triple helix, creating an altered structure that hasbeen shown to strongly provoke the nucleotide excision repair pathwayand to activate the site for recombination with the donoroligonucleotide. Two PNA strands can also be linked together to form abis-PNA molecule.

The triplex-forming molecules are useful to induce site-specifichomologous recombination in mammalian cells when used in combinationwith one or more donor oligonucleotides which provides the correctedsequence. Donor oligonucleotides can be tethered to triplex-formingmolecules or can be separate from the triplex-forming molecules. Thedonor oligonucleotides can contain at least one nucleotide mutation,insertion or deletion relative to the target duplex DNA.

Double duplex-forming molecules, such as a pair of pseudocomplementaryoligonucleotides, can also induce recombination with a donoroligonucleotide at a chromosomal site. Use of pseudocomplementaryoligonucleotides in targeted gene therapy is described in U.S. PublishedApplication No. 2011/0262406. Pseudocomplementary oligonucleotides arecomplementary oligonucleotides that contain one or more modificationssuch that they do not recognize or hybridize to each other, for exampledue to steric hindrance, but each can recognize and hybridize tocomplementary nucleic acid strands at the target site. In someembodiments, pseudocomplementary oligonucleotides are pseudocomplemenarypeptide nucleic acids (pcPNAs). Pseudocomplementary oligonucleotides canbe more efficient and provide increased target site flexibility overmethods of induced recombination such as triple-helix oligonucleotidesand bis-peptide nucleic acids which require a polypurine sequence in thetarget double-stranded DNA.

B. In Vitro Methods

The disclosed compositions can be used in a method of delivering activeagents to cells in vitro. For example, the nanolipogels can be used forin vitro transfection of cells. The method typically involves contactingthe cells with nanolipogel including a polynucleotide in an effectiveamount to introduce the polynucleotide into the cell's cytoplasm. Insome embodiments, the polynucleotide is delivered to the cell in aneffective amount to change the genotype or a phenotype of the cell. Thecells can primary cells isolated from a subject, or cells of anestablished cell line. The cells can be of a homogenous cell type, orcan be a heterogeneous mixture of different cells types. For example,the polyplexes can be introduced into the cytoplasm of cells from aheterogenous cell line possessing cells of different types, such as in afeeder cell culture, or a mixed culture in various states ofdifferentiation. The cells can be a transformed cell line that can bemaintained indefinitely in cell culture. Exemplary cell lines are thoseavailable from American Type Culture Collection including tumor celllines.

Any eukaryotic cell can be transfected to produce cells that express aspecific nucleic acid, for example a metabolic gene, including primarycells as well as established cell lines. Suitable types of cells includebut are not limited to undifferentiated or partially differentiatedcells including stem cells, totipotent cells, pluripotent cells,embryonic stem cells, inner mass cells, adult stem cells, bone marrowcells, cells from umbilical cord blood, and cells derived from ectoderm,mesoderm, or endoderm. Suitable differentiated cells include somaticcells, neuronal cells, skeletal muscle, smooth muscle, pancreatic cells,liver cells, and cardiac cells. In another embodiment, siRNA, antisensepolynucleotides (including siRNA or antisense polynucleotides) orinhibitory RNA can be transfected into a cell using the compositionsdescribed herein. The methods are particularly useful in the field ofpersonalized therapy, for example, to repair a defective gene,de-differentiate cells, or reprogram cells. For example, target cellsare first isolated from a donor using methods known in the art,contacted with the nanolipogel including a polynucleotide causing achange to the in vitro (ex vivo), and administered to a patient in needthereof. Sources or cells include cells harvested directly from thepatient or an allographic donor. In preferred embodiments, the targetcells to be administered to a subject will be autologous, e.g. derivedfrom the subject, or syngenic. Allogeneic cells can also be isolatedfrom antigenically matched, genetically unrelated donors (identifiedthrough a national registry), or by using target cells obtained orderived from a genetically related sibling or parent.

Cells can be selected by positive and/or negative selection techniques.For example, antibodies binding a particular cell surface protein may beconjugated to magnetic beads and immunogenic procedures utilized torecover the desired cell type. It may be desirable to enrich the targetcells prior to transient transfection. As used herein in the context ofcompositions enriched for a particular target cell, “enriched” indicatesa proportion of a desirable element (e.g. the target cell) which ishigher than that found in the natural source of the cells. A compositionof cells may be enriched over a natural source of the cells by at leastone order of magnitude, preferably two or three orders, and morepreferably 10, 100, 200, or 1000 orders of magnitude. Once target cellshave been isolated, they may be propagated by growing in suitable mediumaccording to established methods known in the art. Established celllines may also be useful in for the methods. The cells can be storedfrozen before transfection, if necessary.

Next the cells are contacted with the disclosed composition in vitro torepair, de-differentiate, re-differentiate, and/or re-program the cell.The cells can be monitored, and the desired cell type can be selectedfor therapeutic administration.

Following repair, de-differentiation, and/or re-differentiation and/orreprogramming, the cells are administered to a patient in need thereof.In the most preferred embodiments, the cells are isolated from andadministered back to the same patient. In alternative embodiments, thecells are isolated from one patient, and administered to a secondpatient. The method can also be used to produce frozen stocks of alteredcells which can be stored long-term, for later use. In one embodiment,fibroblasts, keratinocytes or hematopoietic stem cells are isolated froma patient and repaired, dedifferentiated, or reprogrammed in vitro toprovide therapeutic cells for the patient.

C. Treatment of Inflammatory and Autoimmune Diseases

Chronic and persistent inflammation is a major cause of the pathogenesisand progression of systemic autoimmune diseases such as rheumatoidarthritis (RA) and systemic lupus erythematosus (SLE).

Accordingly, methods of treating inflammatory and autoimmune diseasesand disorders can include administering to a subject in need thereof, aneffective amount of a loaded nanolipogel formulation or a pharmaceuticalcomposition thereof, to reduce or ameliorate one or more symptoms of thedisease or condition.

For example, the nanolipogel can include active agents that facilitateinhibition of cell cycle progression of T cells, for example, activatedT cells (i.e., CD44^(hi) T cells). The reduction can be in activatedperipheral blood CD4+ T cells, splenic CD4+ T cells, or a combinationthereof. In some embodiments, IFN-γ producing T cells are reduced.Preferably there is little or no reduction in regulatory T cells. Insome embodiments there is an increase in regulatory T cells (Tregs).

In some embodiments, the composition is administered in an effectiveamount to induce or increase immunosuppression by modulating immune cellfunction or activation state rather than by lymphodepletion. Forexample, in some embodiments, the composition reduces activation of CD4+T cells, reduces the stimulatory capacity of antigen presenting cellssuch as dendritic cells, or a combination thereof.Lymphodepletion/immune cell depletion may also be achieved withincreases in dosing or with the use of other drugs. In some embodimentsthe composition is administered in an effective amount to increasedifferentiation of CD4+ T cells into Fox3+CD25+ Tregs.

In some embodiments the composition is administered in an effectiveamount to reduce the amount of one or more proinflammatory molecules,the expression of major histocompatibility complex (MHC) I or II, or acombination thereof. Exemplary proinflammatory molecules include, butare not limited to IL-1β, TNF-α, TGF-beta, IFN-γ, IFN-α, IL-17, IL-6,IL-23, IL-22, IL-21, IL-12 and MMPs. In a preferred embodiment, thecomposition is administered in an effective amount to reduce dendriticcell production of TNF-α, IFN-γ, or IL-12, or reduce dendritic cellexpression of MHC I or MHC II, or combinations thereof. In someembodiment CD4+ T cell production of IFN-γ is reduced. In someembodiments, the composition mediates T cell depletion in conjunctionwith the modulation of antigen presenting cells so that they are lesspotent stimulators or become more tolerogenic.

The Examples below show that nanolipogels effectively traffic to immunecells (i.e., in the lymph and spleen, etc.) without a targeting moiety.Accordingly, a targeting moiety is not required to target nanolipogelsto dendritic cells or T cells. However, in some embodiments, a targetingmoiety is added to the nanolipogel particle to increase targeting to aspecific immune cell type, or tissue within the body. For example,depletion of activated CD4-positive T cells, in conjunction with themodulation of antigen presenting cells, such as dendritic cells, so thatthey are less potent stimulators or become tolerogenic, could reduce oneor more disease symptoms or increase the duration of disease remissionby suppressing T cell mediated autoimmunity through two distinct butadditive pathways. Thus it could be advantageous to use a balance ofnanoparticle targeting to both T cells (i.e., activate CD4+ T cells) anddendritic cells.

In some embodiments this is accomplished by co-administering twodifferent particles, one decorated with a targeting moiety that enhancesdelivery to T cells and one with a targeting moiety that enhancesdelivery to antigen presenting cells such as dendritic cells. In oneembodiment, the targeting moiety that directs particles to the CD4 cellsis a CD4 antibody or antigen binding fragment thereof one or a moietyspecifically targeting the T cell receptor. A targeting moiety thatdirects particles to dendritic cells such as by targeting to the DEC-205receptor, any toll-like receptor, cell surface marker or receptor orother C-type lectins on dendritic cells can also be used. The particlescan include one or more active agents. For example, the active agent(s)targeted to T cells can reduce proliferation or activation of T cells,while the active agent(s) can induce dendritic cells to be tolerogenicor suppress their antigen presenting ability or T cell inducingactivity. The two different nanolipogels can be loaded with the same ordifferent active agent(s). In a preferred embodiment, at least one ofthe active agents is mycophenolic acid (MP A), or a derivative oranalogue thereof.

In some embodiments, nanoparticles designed with enhanced penetration tothe white pulp of the secondary lymphoid organs to better inhibit T andB lymphocyte proliferation.

The disclosed nanolipogels typically carry one or more active agentseffective for treating one or more symptoms of an inflammatory orautoimmune disease or disorder for delivery to a cell or tissue in asubject in need thereof. Therapeutic agents include, but are not limitedto, immunosuppressive agents, e.g., antibodies against other lymphocytesurface markers (e.g., CD40) or against cytokines, other fusionproteins, e.g., CTLA4Ig, or other immunosuppressive drugs (e.g.,cyclosporin A, FK506-like compounds, rapamycin compounds, or steroids),anti-proliferatives, cytotoxic agents, or other compounds that mayassist in immunosuppression.

As used herein the term “rapamycin compound” includes the neutraltricyclic compound rapamycin, rapamycin derivatives, rapamycin analogs,and other macrolide compounds which are thought to have the samemechanism of action as rapamycin (e.g., inhibition of cytokinefunction). The language “rapamycin compounds” includes compounds withstructural similarity to rapamycin, e.g., compounds with a similarmacrocyclic structure, which have been modified to enhance theirtherapeutic effectiveness. Exemplary Rapamycin compounds are known inthe art (See, e.g. WO 95122972, WO 95116691, WO 95104738, U.S. Pat. Nos.6,015,809; 5,989,591; U.S. Pat. Nos. 5,567,709; 5,559,112; 5,530,006;5,484,790; 5,385,908; 5,202,332; 5, 162,333; 5,780,462; 5,120,727).

“FK506 compounds” includes FK506, and FK506 derivatives and analogs,e.g., compounds with structural similarity to FK506, e.g., compoundswith a similar macrocyclic structure which have been modified to enhancetheir therapeutic effectiveness. Examples of FK506 compounds include,for example, those described in WO 00101385. The language “rapamycincompound” as used herein does not include FK506-like compounds.

Other suitable therapeutics include, but are not limited to,antiinflammatory agents. The anti-inflammatory agent can benon-steroidal, steroidal, or a combination thereof. One embodimentprovides oral compositions containing about 1% (w/w) to about 5% (w/w),typically about 2.5% (w/w) or an anti-inflammatory agent. Representativeexamples of non-steroidal anti-inflammatory agents include, withoutlimitation, oxicams, such as piroxicam, isoxicam, tenoxicam, sudoxicam;salicylates, such as aspirin, disalcid, benorylate, trilisate, safapryn,solprin, diflunisal, and fendosal; acetic acid derivatives, such asdiclofenac, fenclofenac, indomethacin, sulindac, tolmetin, isoxepac,furofenac, tiopinac, zidometacin, acematacin, fentiazac, zomepirac,clindanac, oxepinac, felbinac, and ketorolac; fenamates, such asmefenamic, meclofenamic, flufenamic, niflumic, and tolfenamic acids;propionic acid derivatives, such as ibuprofen, naproxen, benoxaprofen,flurbiprofen, ketoprofen, fenoprofen, fenbufen, indopropfen, pirprofen,carprofen, oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen,alminoprofen, and tiaprofenic; pyrazoles, such as phenylbutazone,oxyphenbutazone, feprazone, azapropazone, and trimethazone. Mixtures ofthese non-steroidal anti-inflammatory agents may also be employed.

Representative examples of steroidal anti-inflammatory drugs include,without limitation, corticosteroids such as hydrocortisone,hydroxyl-triamcinolone, alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasone dipropionates, clobetasol valerate,desonide, desoxymethasone, desoxycorticosterone acetate, dexamethasone,dichlorisone, diflorasone diacetate, diflucortolone valerate,fluadrenolone, fluclorolone acetonide, fludrocortisone, flumethasonepivalate, fluosinolone acetonide, fluocinonide, flucortine butylesters,fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone,halcinonide, hydrocortisone acetate, hydrocortisone butyrate,methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone,flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone,fludrocortisone, diflurosone diacetate, fluradrenolone acetonide,medrysone, amcinafel, amcinafide, betamethasone and the balance of itsesters, chloroprednisone, chlorprednisone acetate, clocortelone,clescinolone, dichlorisone, diflurprednate, flucloronide, flunisolide,fluoromethalone, fluperolone, fluprednisolone, hydrocortisone valerate,hydrocortisone cyclopentylpropionate, hydrocortamate, meprednisone,paramethasone, prednisolone, prednisone, beclomethasone dipropionate,triamcinolone, and mixtures thereof.

In some embodiments, the active agent is an anti-inflammatory cytokineor chemokine, for example, interleukin (IL)-1 receptor antagonist, IL-4,IL-6, IL-10, IL-11, and IL-13. Specific cytokine receptors for IL-1,tumor necrosis factor-alpha, and IL-18 also function as pro-inflammatorycytokine inhibitors. The nature of anti-inflammatory cytokines andsoluble cytokine receptors are known in the art and discussed in Opaland DePalo, Chest, 117(4): 1162-72 (2000).

Retinoic acid is an additional therapeutic compound that could be usedas an antinflammatory agent. See, for example, Capurso, et a.Self/Nonself, 1:4, 335-340 (2010).

Alternatively, antigens/epitopes specific to an autoimmune condition forwhich tolerance is lost may be added for antigen-specificimmunosuppression.

As discussed in more detail below and in the Examples, a preferredactive agent is MPA.

1. Autoimmune Disease

The autoimmune disease systemic lupus erythematosus (SLE) ischaracterized by multi-organ damage that is caused by T and B cells thatpromote autoantibody production and innate immune cells that mediateinflammation. The ability to target and inactivate these immune cellswith immunosuppressive drugs is a much sought after modality for lupustherapies and autoimmune disease in general.

The examples demonstrate that nanogels loaded with mycophenolic acid(MPA), a commonly used immunosuppressant drug, extended the survival oflupus-prone NZB/W F1 mice by 12 weeks compared to an equivalent dose offree drug. A 16-fold greater dose of MPA administered in buffer couldnot achieve the same therapeutic effect as MPA-loaded nanogels.Strikingly, the nanogel mechanism of therapy was dependent on efficienttrafficking to lymphoid organs, and their association with CD4 T cellsas well as conventional and plasmacytoid dendritic cells. Cells whichhave bound or internalized nanogels were shown to be less proliferativeand have reduced production of inflammatory cytokines. The resultsreveal for the first time a comprehensive study in the potential use ofnanoparticles for lupus therapy and implicate a mechanism for enhancingtherapeutic immunosuppression in autoimmune disease by targeting both Tcells and antigen-presenting cells.

Strikingly, the nanogel mechanism of therapy was dependent on efficienttrafficking to lymphoid organs. Nanolipogels associated with CD4 T cellsas well as conventional and plasmacytoid dendritic cells. Cells whichhave bound or internalized nanogels were shown to be less proliferativeand have reduced production of inflammatory cytokines.

Accordingly, in a preferred embodiment, the nanolipogel is used todeliver MPA to a CD4 T cells, dendritic cells, or a combination thereofin an effective amount to a reduce one or more symptoms of SLE.

Mesangial cells in the kidney promote the glomerular damage associatedwith lupus mortality, and methods that inhibit these cells represent analternative, and potentially synergistic, mechanism of therapy.

Accordingly, in some embodiments, nanolipogels carrying an active agentthat inhibits mesangial cells, for example MPA, are targeted to kidney.In some embodiments, the compositions reduce one or more symptoms ofSLE. For example, the composition can prevent, delay, or reduce theseverity of proteinuria; reduce the production of anti-nuclearautoantibodies (ANA); reduce abnormal lympoproliferation; prevent, delayor reduce glomerular nephritis; reduce, prevent or delay elevated bloodurea levels; or combinations thereof. Treatment is similar for othertypes of autoimmune disease such as psoriasis and rheumatoid arthritis.

2. Allergies

A similar methodology can be used to treat allergies, substituting theallergen of interest for the autoimmune stimulus.

Allergies are abnormal reactions of the immune system that occur inresponse to otherwise harmless substances. Allergies are among the mostcommon of medical disorders. It is estimated that 60 million Americans,or more than one in every five people, suffer from some form of allergy,with similar proportions throughout much of the rest of the world.Allergy is the single largest reason for school absence and is a majorsource of lost productivity in the workplace.

An allergy is a type of immune reaction. Normally, the immune systemresponds to foreign microorganisms or particles by producing specificproteins called antibodies. These antibodies are capable of binding toidentifying molecules, or antigens, on the foreign particle. Thisreaction between antibody and antigen sets off a series of chemicalreactions designed to protect the body from infection. Sometimes, thissame series of reactions is triggered by harmless, everyday substancessuch as pollen, dust, and animal danders. When this occurs, an allergydevelops against the offending substance (an allergen.) Mast cells, oneof the major players in allergic reactions, capture and display aparticular type of antibody, called immunoglobulin type E (IgE) thatbinds to allergens. Inside mast cells are small chemical-filled packetscalled granules. Granules contain a variety of potent chemicals,including histamine. Immunologists separate allergic reactions into twomain types: immediate hypersensitivity reactions, which arepredominantly mast cell-mediated and occur within minutes of contactwith allergen; and delayed hypersensitivity reactions, mediated by Tcells (a type of white blood cells) and occurring hours to days afterexposure.

Inhaled or ingested allergens usually cause immediate hypersensitivityreactions. Allergens bind to IgE antibodies on the surface of mastcells, which spill the contents of their granules out onto neighboringcells, including blood vessels and nerve cells. Histamine binds to thesurfaces of these other cells through special proteins called histaminereceptors.

Interaction of histamine with, receptors on blood vessels causesincreased leakiness, leading to the fluid collection, swelling andincreased redness. Histamine also stimulates pain receptors, makingtissue more sensitive and irritable. Symptoms last from one to severalhours following contact. In the upper airways and eyes, immediatehyper-sensitivity reactions cause the runny nose and itchy, bloodshoteyes typical of allergic rhinitis. In the gastrointestinal tract, thesereactions lead to swelling and irritation of the intestinal lining,which causes the cramping and diarrhea typical of food allergy.Allergens that enter the circulation may cause hives, angioedema,anaphylaxis, or atopic dermatitis.

Allergens on the skin usually cause delayed hypersensitivity reaction.Roving T cells contact the allergen, setting in motion a more prolongedimmune response. This type of allergic response may develop over severaldays following contact with the allergen, and symptoms may persist for aweek or more.

Allergens enter the body through four main routes: the airways, theskin, the gastrointestinal tract, and the circulatory system. Airborneallergens cause the sneezing, runny nose, and itchy, bloodshot eyes ofhay fever (allergic rhinitis). Airborne allergens can also affect thelining of the lungs, causing asthma, or conjunctivitis (pink eye).Exposure to cockroach allergens has been associated with the developmentof asthma. Airborne allergens from household pets are another commonsource of environmental exposure. Allergens in food can cause itchingand swelling of the lips and throat, cramps, and diarrhea. When absorbedinto the bloodstream, they may cause hives (urticaria) or more severereactions involving recurrent, noninflammatory swelling of the skin,mucous membranes, organs, and brain (angioedema). Some food allergensmay cause anaphylaxis, a potentially life-threatening condition markedby tissue swelling, airway constriction, and drop in blood pressure.Allergies to foods such as cow's milk, eggs, nuts, fish, and legumes(peanuts and soybeans) are common. Allergies to fruits and vegetablesmay also occur. In contact with the skin, allergens can cause reddening,itching, and blistering, called contact dermatitis. Skin reactions canalso occur from allergens introduced through the airways orgastrointestinal tract. This type of reaction is known as atopicdermatitis. Dermatitis may arise from an allergic Dermatitis may arisefrom an allergic response (such as from poison ivy), or exposure to anirritant causing nonimmune damage to skin cells (such as soap, cold, andchemical agents). Injection of allergens, from insect bites and stingsor drug administration, can introduce allergens directly into thecirculation, where they may cause system-wide responses (includinganaphylaxis), as well as the local ones of swelling and irritation atthe injection site.

3. Other Inflammatory and Autoimmune Disease and Disorders

The results in the Examples below demonstrate the use of nanoparticlesfor lupus therapy and illustrate a mechanism that can be used fortherapeutic immunosuppression strategies useful in the treatment ofinflammatory diseases or disorders, autoimmune diseases or disorders,inducing or increase graft tolerance, treating graft rejection, andtreating allergies and other alignments with symptoms that can bereduced or ameliorated by regulating the activity of T cells,antigen-presenting cells, or combinations thereof.

Representative inflammatory responses or autoimmune diseases that can bedetected or assessed for severity include, but are not limited to,rheumatoid arthritis, systemic lupus erythematosus, alopecia areata,anklosing spondylitis, antiphospholipid syndrome, autoimmune Addison'sdisease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmuneinner ear disease, autoimmune lymphoproliferative syndrome (alps),autoimmune thrombocytopenic purpura (ATP), Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatiguesyndrome immune deficiency, syndrome (CFIDS), chronic inflammatorydemyelinating polyneuropathy, cicatricial pemphigoid, cold agglutinindisease, Crest syndrome, Crohn's disease, Dego's disease,dermatomyositis, dermatomyositis—juvenile, discoid lupus, essentialmixed cryoglobulinemia, fibromyalgia—fibromyositis, grave's disease,guillain-barre, hashimoto's thyroiditis, idiopathic pulmonary fibrosis,idiopathic thrombocytopenia purpura (ITP), Iga nephropathy, insulindependent diabetes (Type I), juvenile arthritis, Meniere's disease,mixed connective tissue disease, multiple sclerosis, myasthenia gravis,pemphigus vulgaris, pernicious anemia, polyarteritis nodosa,polychondritis, polyglancular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome,rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-mansyndrome, Takayasu arteritis, temporal arteritis/giant cell arteritis,ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener'sgranulomatosis.

Graft Tolerance

These results are also indicative of efficacy in treating graftrejection and allergies. For example, the compositions can be used inthe maintenance of transplants, in which drug combinations are desirableto prevent rejection. The transplanted material can be cells, tissues,organs, limbs, digits or a portion of the body, preferably the humanbody. The transplants are typically allogenic or xenogenic. Nanolipogelsare administered to a subject in an effective amount to reduce orinhibit transplant rejection. Nanolipogels can be administeredsystemically or locally by any acceptable route of administration. Insome embodiments, the nanolipogel particles are administered to a siteof transplantation prior to, at the time of, or followingtransplantation. In one embodiment, the nanolipogel particles areadministered to a site of transplantation parenterally, such as bysubcutaneous injection.

In other embodiments nanolipogel particles are administered directly tocells, tissue or organ to be transplanted ex vivo. In one embodiment,the transplant material is contacted with nanolipogel particles prior totransplantation, after transplantion, or both.

In other embodiments nanolipogel particles are administered to immunetissues or organs, such as lymph nodes or the spleen.

The transplant material can be treated with enzymes or other materialsthat remove cell surface proteins, carbohydrates, or lipids that areknown or suspected in being involved with immune responses such astransplant rejection.

a. Cells

Populations of any types of cells can be transplanted into a subject.The cells can be homogenous or heterogeneous. Heterogeneous means thecell population contains more than one type of cell. Exemplary cellsinclude progenitor cells such as stem cells and pluripotent cells whichcan be harvested from a donor and transplanted into a subject. The cellsare optionally treated prior to transplantation as mention above.

Ex vivo methods of nucleic acid delivery can include, for example, thesteps of harvesting cells from a subject, culturing the cells,transducing them with an expression vector using nanolipogel particles,and maintaining the cells under conditions suitable for expression ofthe encoded polypeptides. These methods are known in the art ofmolecular biology.

b. Tissues

Any tissue can be used as a transplant. Exemplary tissues include skin,adipose tissue, cardiovascular tissue such as veins, arteries,capularies, valves; neural tissue, bone marrow, pulmonary tissue, oculartissue such as corneas and lens, cartilage, bone, and mucosal tissue.The tissue can be modified as discussed above.

c. Organs

Exemplary organs that can be used for transplant include, but are notlimited to kidney, liver, heart, spleen, bladder, lung, stomach, eye,tongue, pancreas, intestine, etc. The organ to be transplanted can alsobe modified prior to transplantation as discussed above.

One embodiment provides a method of inhibiting or reducing chronictransplant rejection in a subject by administering an effective amountof nanolipogel particles to inhibit or reduce chronic transplantrejection relative to a control.

5. Graft-Versus-Host Disease (GVHD)

Nanolipogel particles can also be used to treat graft-versus-hostdisease (GVHD) by administering an effective amount of nanolipogelparticles including an active agent to alleviate one or more symptomsassociated with GVHD. GVHD is a major complication associated withallogeneic hematopoietic stem cell transplantation in which functionalimmune cells in the transplanted marrow recognize the recipient as“foreign” and mount an immunologic attack. It can also take place in ablood transfusion under certain circumstances. Symptoms of GVD includeskin rash or change in skin color or texture, diarrhea, nausea, abnormalliver function, yellowing of the skin, increased susceptibility toinfection, dry, irritated eyes, and sensitive or dry mouth.

6. Diabetes

The nanolipogel particles can also be used to treat diabetes. The methodincludes transplanting insulin producing cells in a subject andadministering to the subject an effective amount of nanolipogelparticles including an active agent to reduce or inhibit transplantrejection. In another method, the pancreatic islet antigens can beencapsulated together with a tolerogenic agent and used as to inducetolerance against the insulin producing cells. Preferably the insulinproducing cells are beta cells or islet cells. In certain embodiments,the insulin producing cells are recombinant cells engineered to produceinsulin.

The insulin producing cells can be encapsulated within a matrix, such asa polymeric matrix, using suitable polymers, including, but not limitedto alginate, agarose, hyaluronic acid, collagen, synthetic monomers,albumin, fibrinogen, fibronectin, vitronectin, laminin, dextran, dextransulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, chitin,chitosan, heparan, heparan sulfate, or a combination thereof.

D. Exemplary Disease Treatment Strategies

Exemplary therapies and strategies of autoimmune diseases and otherinflammatory conditions are outlined in Table 1 below. The tablepresents a pathological aberrance that is addressed by the therapy; thecells target(s) of the therapy; one, two, or three therapeutic moleculesthat can be delivered by the nanolipogels alone or in any combinationthereof; a desired target or targeting moiety can be used to target thenanoliposomes; the preferred delivery mechanism; and intended effects ofthe therapy.

TABLE 1 Exemplary Therapies and Strategies for Treating Inflammatory andAutoimmune Diseases and Disorders Th2- Immune committed Inflammatorysystem is cells are response T cells Parasite Parasite reactive hijackedby Pathological against self reacting infects lives in against differentTh17 Aberrance Ag against graft RBCs macrophages collagen pathogenscells Cell Antigen T cells Infected Infected APCs At-risk T Th17Target(s) Presenting (naïve) RBCs macrophages cells cells Cells (APCs)Delivered Plasmid for Foxp3 Plasmid Myriad small HDACi HDACi for HDACiMolecule tolerogenic plasmid DNA molecule prescribed against (DM) 1cytokine drugs transcription outlined such as IL-10 factors targets DM2Self-antigen Tolerogenic More Excess Collagen siRNA (collagen, drug likeplasmid dendrimer to autoantigen against insulin, etc) rapamycin DNAhelp break outlined endosome targets DM3 Protein, Maybe peptide,tolerogenic something drug Ag-specific Targeting Anti-CD7 for Mannose,Fc Strategy human fragment, etc Delivery Choukri Mechanism Mamounplasmids Effect Create Change Transform Expel parasite Abrogate antigen-inflammatory parasite, from autoimmune specific T cells to identifyendolysosome, environment tolerance Ag-specific drug kill it APCs thattolerogenic resistance amplify type infected tolerance RBCs

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLES Example 1: Preparation of Nanolipogels for Delivery ofAnti-Tumor Molecules

Materials and Methods

Nanolipogel synthesis. “Nanolipogel” (sometimes referred to as “nLG”)particles due to its nature as a lipid bilayer surrounding a hydrogelcore was fabricated from a degradable polymer (FIG. 1B). Liposomes wereused as nanoscale molds for photo-initiated hydrogel formation. Toachieve sustained release of the hydrophobic drug in conjunction withencapsulated proteins, methacrylate-conjugated β-cyclodextrins (CDs)were incorporated into the interior of the liposomes, β-cyclodextrinshave a long history as solublization agents for hydrophobic compoundsand are key excipients in various pharmaceutical formulations. Thisformulation procedure enabled coencapsulation of both proteins as wellas small hydrophobic drugs within the interior of the lipid bilayer(FIG. 1A-1B).

Conjugated CDs were created by reaction of succinylated-CDs withphotosensitive methacrylate groups through hydro lysable ester groups.(FIG. 1A) Complexation of SB or rhodamine (for imaging) withfunctionalized CD was verified by proton nuclear magnetic resonance (1HNMR) on a 500 MHz Bruker spectrometer. All samples were dissolved in1-10 mg/ml in D₂O for characterization with the solvent as a reference.

PLA-PEG-PLA diacrylate was synthesized in two steps according toSawhney, et al. Macromole 26, 581-587 (1993). All chemicals werepurchased from Sigma unless otherwise noted and were of ACS grade orhigher, α,ω-dihydroxy poly(ethylene oxide) with a molecular weight of4000 g/mol, 3,6-dimethyl-1,4-dioxane-2,5-dione (dl-lactide), and tin(II)2-ethylhexanoate (stannous octoate) were charged into a round-bottomflask under nitrogen in a 5:1:0.0075 mol ratio and the reaction wasstirred under vacuum at 200° C. for 4 hours, followed by stirring at160° C. for 2 hours. After cooling to room temperature, the resultingcopolymer was dissolved in dichloromethane and precipitated in anhydrousether. This intermediate was dissolved in dichloromethane (10 g/mL) andcooled to 0° C. in an ice bath. Per 10 g of polymer intermediate, 440triethylamine and 530 acryloyl chloride were added under nitrogen andthe reaction mixture was stirred for 12 hours at 0° C. and 12 hours atroom temperature. The mixture was filtered and the resulting polymer wasprecipitated in diethyl ether. The final polymer was redissolved indichloromethane, re-precipitated in hexanes and characterized by FTIRand NMR.

The complexation of Rhodamine and SB505124 with cyclodextrins wasexamined by proton nuclear magnetic resonance (¹H NMR) spectroscopy on a500 MHz Bruker spectrometer.

Nanolipogel formulation. All lipids were obtained from Avanti PolarLipids and used without further preparation. Phosphatidyl choline (PC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(poly ethyleneglycol)-2000] (DSPE-PEG), and cholesterol were mixed in chloroform in a3:1:1 molar ratio and liposomes were formulated using a remote loadingtechnique of Peer, et al. Science 319, 627-630 (2008). Lipid-labeledfluorescent liposomes were formulated by incorporation of 10%1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethyleneglycol)2000-N′-carboxyfluorescein] (DSPE-PEG-Fluorescein). Briefly, thedissolved lipids were mixed in a glass scintillation vial, followed bycomplete solvent removal with a directed nitrogen stream. This formed athin lipid film on the inner glass surfaces, which was rehydrated by theaddition of IX phosphate buffered saline (PBS). Cycles of thirty secondvortexing followed by 5 min idle sitting at room temperature wererepeated ten times and the resulting multilamellar liposomes wereextruded 10 times through a 5 μm polycarbonate membrane (Whatman), 10times through a 1 μm membrane and finally 11 times through a 100 nmusing a LIPEX extruder (Northern Lipids, Inc.). The resultingunilamellar liposomes were then frozen and lyophilized.

Lyophilized liposomes were reconstituted with a solution containing 5%(w/v) polymer (FIG. 1B) and 2.5 mg/mL Ciba Irgacure 2959 as thephotoinitiator and: no other additive (nLG-Empty), 9 mg f-CD-solubilizedSB/100 mg nLG (nLG-SB; SB505124, Sigma), 1 μg IL-2/100 mg lipids(LG-IL-2; Aldesleukin Proleukin, Novartis), or both f-CD-solubilized SBand IL-2 (nLG-SB+IL-2). CD (randomly succinylated β-CD; CTD, Inc.) wasfunctionalized with 2-aminoethyl methacrylate by stirring a 1:3 molarratio of the compounds in IX PBS for 1 hour at room temperature. SB wasincorporated into f-CD by adding the drug dissolved in methanol to thef-CD. After 20 minutes of vigorous stirring at room temperature to formthe complexes, the methanol was evaporated with a directed stream ofnitrogen. The reconstitution step proceeded with 30 minutes of vortexingto rehydrate the liposomes. The liposomes were then irradiated under UVlight for 8 minutes with a Blak-Ray long wave ultraviolet lamp (Model B100) at a 10 cm working distance.

Directly prior to UV irradiation, the samples were diluted fivefold toprevent macroscale gellation. The resulting nanolipogels were pelletedby centrifugation (five minutes at 7200 rcf) and resuspended in IX PBS.This centrifugation/resuspension procedure was repeated three times.Nanolipogels were aliquotted and frozen at −20° C. until further use.For consistency, all nanolipogels were frozen prior to use (in vitro orin vivo). Final size and dispersity was confirmed by resuspendingnanolipogels in IX PBS for analysis on a ZetaPALS dynamic lightscattering instrument. The zeta potential of PC/cholesterol liposomes,PC/cholesterol/PE-PEG-NH₂ liposomes, and nanolipogels were evaluated in0.1×PBS using a Malvern nanosizer. For TEM analysis, nanolipogel sampleswere stained with osmium tetroxide and then imaged on an FEI TenaiBiotwin microscope. Lipid-specific osmium tetroxide staining ofcryosectioned samples had a localized staining pattern confined to theexterior membrane of the particle.

Results

Liposomes were used as nanoscale molds for photo-initiated hydrogelformation. To achieve sustained release of the hydrophobic drug inconjunction with encapsulated proteins, methacrylate-conjugatedβ-cyclodextrins (CDs) were incorporated into the interior of theliposomes, β-cyclodextrins have a long history as solublization agentsfor hydrophobic compounds and are key excipients in variouspharmaceutical formulations.

Complexation of SB or rhodamine (for imaging) with functionalized CD wasverified using ¹H NMR. The functionalized CD (f-CD) becomes covalentlybound to the liposome-encapsulated polymer matrix during photo-inducedpolymerization, thus the SB can only be released upon f-CD/SB hydrolysisof the polymer ester groups and subsequent diffusion out of thenanolipogel, enabling sustained release compared to the burst-dominatedrelease of SB in the absence of gelled CD. This system enabled controlover the release of remotely loaded IL-2 without compromising itsbioactivity and enabled simultaneous release of both protein and drugcompared to single component release. The release profile ofSB/IL-2-loaded nanolipogels was not altered by incubation in serum andrelease was substantively completed by 7 days.

To demonstrate the impact of polymerization in the nLG on the releaseprofile of SB and IL-2, release kinetics of both agents were comparedwith release from liposomes and solid poly(lactide-co-glycolide)nanoparticles (PLGA NPs) encapsulating both agents. Incorporation ofphotocured polymer in the nanolipogel vehicle enabled a more sustainedrelease of SB compared to liposomes and a more complete release comparedto conventional 50:50 (PLGA NPs) of the same diameter. The releasekinetics of the drug is seen to be intermediate between that ofdiffusion dependent release from liposomes and hydrolysis dependentrelease from PLGA. Comparative cumulative release of IL-2 fromliposomes, nanolipogels, and PLGA NPs demonstrated that encapsulation ofIL-2 in nanolipogels enabled better sustained release of cytokine.

The bioactivity of the SB and IL-2 were unaffected by lipogelincorporation. Encapsulation of IL-2 (80%) and/or drug (36%) did notsignificantly affect nanolipogel diameter; dynamic light scatteringanalysis revealed a mean diameter of 120 nm and polydispersity index of0.2. Liposomes and nanolipogels incorporating amine-terminated PEGylatedphosphatidyl ethanolamine demonstrated a neutral zeta potential,compared to the approximately −22±10 mV zeta potential of liposomesformulated with only phosphatidyl choline and cholesterol. Cryo-TEM ofnanolipogels showed the formation of spherical liposomal structures,detectable by light scattering even after disruption of the liposomalexterior by detergent, validating an inner gel core with approximatelythe same diameter as the intact nanolipogel. The in vitro cytotoxicityof this system was negligible.

To investigate the biodistribution and clearance of this platform,CD-solubilized rhodamine was used as a fluorescent surrogate markermodel for SB; rhodamine complexation with CD had been previously used toqualify guest-host interactions with CDs. This was confirmed here by ¹HNMR. Encapsulation of SB or SB+IL-2 had no significant effect onparticle mean diameter or polydispersity. FIG. 1F shows that the zetapotential of liposomes and nanolipogels incorporating amine-terminatedPE-PEG was found to be close to neutral. FIG. 1G shows the compositionand formulation properties of the nanolipogel formulation. FIG. 1H showsthe polymer structure verified by ¹H NMR. Cryo-TEM of nanolipogelsdemonstrated the formation of spherical liposomal structures. FIG. 1Ishows that the photopolymerized polymer/CD forms nanoparticulatehydrogel structures that are detectable by light scattering even afterdisruption of the liposomal exterior by detergent.

Example 2: In Vitro Release and Bioactivity Studies

Materials and Methods

Controlled release studies. To demonstrate the advantage of nanolipogelvehicles for controlled release of encapsulated agents over prolongedperiods of time, a series of studies were conducted to evaluate in vitrorelease of nanolipogel particles containing SB and/or IL-2. Releasestudies were performed at 37° C. with constant agitation in IX PBS+10%fetal bovine serum. At each time point the complete volume was removedand replaced with fresh buffer after centrifugation (five minutes at7200 ref). Nanolipogels were resuspended by manual pipetting. Absorbancemeasurements to determine SB concentrations were performed with aBeckman Coulter plate reader at 300 nm. Absorbance readings fromnLG-Empty particles were subtracted from those obtained from nLG-SBparticles to ensure readings were due only to encapsulated SB. IL-2release was determined using an IL-2 ELISA kit (BD Biosciences) withhumanized capture (BD, 555051) and biotinylated-detection (BD, 555040)antibodies according to the manufacturer's instructions. For IL-2 usedin these studies, the international unit conversion was 22 MU=1.3 mg.

The functionalized CD (f-CD) becomes covalently bound to theliposome-encapsulated polymer matrix during photo-inducedpolymerization, thus the SB can only be released upon f-CD/SB hydrolysisof the polymer ester groups and subsequent diffusion out of thenanolipogel, enabling sustained release compared to the burst-dominatedrelease of SB in the absence of gelled CD.

Bioactivity studies. Cumulative release of nLG-IL-2 was performed at 1,3, 5, and 7 days in complete media [RPMI media (Gibco) with 10% fetalbovine serum (Atlanta Biological) and penicillin/streptomycin (Sigma)supplemented with L-glutamine (Sigma), non-essential amino acids(Gibco), Hepes buffer (Sigma), gentamicin (Sigma), and β-mercaptoethanol(Sigma)]. Splenocytes were isolated from a B6 mouse and 1×10⁶ cells wereadded in 500 μL T cell media to each well of a 24 well plate previouslycoated with 10 μg/mL anti-CD3 (coated overnight in IX PBS at 4° C.) and5 μg/mL soluble anti-CD28 (BD Biosciences). The media from releasestudies was filtered through a 0.22-μm syringe filter (Whatman) and 500μL was added to the wells. Additionally all wells contained 5 μg/mLsoluble anti-CD28 (BD Biosciences). Soluble IL-2 was added at varyingconcentrations to control wells to as a standard. Cells were incubatedat 37° C. and cellular stimulation was assessed after 72 hours using anIFN-γ ELISA (BD Biosciences).

Results

FIGS. 2A-2E are comparative release profiles from nLG, lipsomes andsolid polymer nanoparticles (PLGA). Cumulative CD- or methacrylatefunctionalized-CD (f-CD)-solubilized SB released from nLGs normalized byinitial carrier mass demonstrated that polymerization of nanolipogelsimproved the sustained nature of SB release (FIG. 2A). Hydroxypropylβ-CD was used for SB complexation with the unfunctionalized CD.Cumulative IL-2 released determined by ELISA (immunoactive) and by abioactivity study (bioactive) from nLGs normalized by initialnanolipogel mass demonstrated that bioactivity of IL-2 was unaffected byencapsulation (FIG. 2B). Release of SB and IL-2 was not affected byincubation of 10 mg nLG in 1 ml full serum (FIG. 2C). Comparativecumulative release of SB from liposomes, nanolipogels, and degradablepolymeric (poly lactide-co-glycolide) nanoparticles (PLGA NPs)demonstrated that incorporation of photo-cured polymer in thenanolipogel vehicle enabled better sustained release and more completerelease of cyclodextrin-solubilized SB (FIG. 2D). PLGA NPs (meandiameter=150+50 nm) were prepared by using a modified water/oil/waterdouble emulsion technique. Liposomes were prepared in an identicalmanner as the nLG without the polymer core. Liposomes were loaded withIL-2 and SB similar to nanolipogels. The diminished percent ofencapsulated SB released from PLGA NPs is attributed to the interactionof the relatively hydrophobic polymer with SB. All particulateformulations were dissolved in 0.1N NaOH+1% SDS to determine 100%release at 7 days (arrow) (FIG. 2D). Comparative cumulative release ofIL-2 from liposomes, nanolipogels, and PLGA NPs demonstrated thatencapsulation of IL-2 in nanolipogels enabled better sustained releaseof cytokine. Cumulative release is presented as % of total IL-2 releasedthrough 7 days. (FIG. 2E) Data in all graphs represent mean oftriplicate samples±1 standard deviation. FIG. 2F compares the sizes andloading of IL-2 and SB in PLGA, nanolipogels and liposomes.

This system enabled control over the release of remotely loaded IL-2without compromising its bioactivity. Loading of IL-2 in the polymerhydrogel space outside of the CD enabled simultaneous release of bothprotein and drug. The decreased total release of both components (FIG.2C) compared to single component release was likely due to stericlimitations within the interior of the nanolipogel or decreased loadingefficiency of SB and IL-2. The release profile of SB/IL-2-loadednanolipogels was not altered by incubation in serum and release wassubstantively completed by 7 days.

To demonstrate the impact of polymerization in the nanolipogel on therelease profile of SB and IL-2 the release kinetics of both agents werecompared with release from liposomes and solidpoly(lactide-co-glycolide) nanoparticles (PLGA NPs) encapsulating bothagents. Incorporation of photocured polymer in the nanolipogel vehicleenabled a more sustained release of SB compared to liposomes and a morecomplete release compared to conventional 50:50 (PLGA NPs) of the samediameter. The release kinetics of the drug is seen to be intermediatebetween that of diffusion-dependent release from liposomes andhydrolysis-dependent release from PLGA. Comparative cumulative releaseof IL-2 from liposomes, nanolipogels, and PLGA NPs demonstrated thatencapsulation of IL-2 in nanolipogels enabled better sustained releaseof cytokine.

Bioactivity.

Nanolipogel vehicles provide the wherewithal to control release ofencapsulated agents without compromising bioactivity. The bioactivity ofthe SB and IL-2 were unaffected by lipogel incorporation. IFN-γproduction was correlated with IL-2 concentration to determinebioactivity.

Example 3: Characterization of Nanolipogels

Encapsulation of IL-2 (80%) and/or drug (36%) did not significantlyaffect nanolipogel diameter; dynamic light scattering analysis revealeda mean diameter of 120 nm and polydispersity index of 0.2. Liposomes andnanolipogels incorporating amine-terminated PEGylated phosphatidylethanolamine demonstrated a neutral zeta potential, compared to the−22±10 mV zeta potential of liposomes formulated with only phosphatidylcholine and cholesterol. Cryo-TEM of nanolipogels showed the formationof spherical liposomal structures, detectable by light scattering evenafter disruption of the liposomal exterior by detergent, validating aninner gel core with approximately the same diameter as the intactnanolipogel. The in vitro cytotoxicity of this system was negligible.

Example 4: Biodistribution

To investigate the biodistribution and clearance of this platform,CD-solubilized rhodamine was as a fluorescent surrogate marker model forSB; rhodamine complexation with CD had been previously used to qualifyguest-host interactions with CDs. This was confirmed by ¹H NMR. The invivo pharmacokinetics of rhodamine following systemic administration wasevaluated in healthy mice receiving a single intravenous administrationof nLG-rhod, an equivalent dose of free rhodamine, or PBS control viatail vein injection.

Results

Spectrofluorometric analysis of rhodamine extracted from blood showed15.7±4.1% and 7.7±3.7% (mean±s.d.) of the initial dose of nanolipogelremaining at 1 and 24 hours respectively post-injection. Free rhodaminewas rapidly cleared and was not detectable in blood at any of the timepoints taken following injection.

FIGS. 3A-3H are graphs showing controlled release, clearance, andbiodistribution. The distribution of both nanolipogel carrier andencapsulated drug payload was investigated using dual-labeled NLG;fluorescein-labeled phosphoethanolamine was incorporated into the lipidcomponent of rhodamine-loaded nanolipogels. Spectrofluorimetric analysisat 540/625 nm and 490/517 nm show dose-dependent fluorescence with nospectral overlap. FIG. 3A is a graph of cumulative IL-2 (ng/mg nLG) anddrug (μ g SB/mg nLG) released from co-loaded nLGs normalized by carriermass. Error bars in all plots represent ±1 standard deviation. FIG. 3Bis a graph showing clearance (percent of initial dose) of drug dose overtime in days: Encapsulation in nanolipogels significantly increased theremaining percentage of initial dose in the blood at 1 and 24 hourspost-injection (two population t test, p<0.01 ###). FIGS. 3C and 3D aregraphs showing whole body distribution. Mice received a single dose ofrhodamine-loaded nanolipogel (FIG. 3C) or soluble rhodamine (in saline)(FIG. 3D) via intravenous tail vein injection. Animals were sacrificedat 1, 24, 48, and 72 hours post-injection for extraction andquantification of fluorescence.

Whole body biodistribution was determined with rhodamine labeling.Significantly higher (two population t test, p<0.01) amounts ofrhodamine were detected in the major organs of nanolipogel-treatedanimals compared to animals injected with free dye. FIG. 3E is a graphof time dependent accumulation in subcutaneous tumor, showing cumulativerhodamine tumor penetration (circles) after B16 peritumoral injection inB6 mice. Peritumoral tissue was collected to quantify the remaining doseof nLG surrounding the tumor (squares).

Controlled release demonstrates release of rhodamine, but not lipid(FIG. 3F). Mice bearing subcutaneous B16 tumors received a single IV(tail vein) injection of dual-labeled NLG. Animals were sacrificed at 1,2, 3, and 7 days post injection and tissues collected forhomogenization, extraction, and quantification of rhodamine andfluorescein-PE. Analysis of serum showing prolonged circulation of bothencapsulant and delivery vehicle. Similar patterns of biodistributionwere observed between lipid (FIG. 3G) and drug payload (FIG. 3H), withhighest accumulations of drug occurring in the lungs and liver.

Analysis of the biodistribution to major organs showed that the lungs,liver and kidney were primary sites of accumulation of bothnanolipogel-encapsulated rhodamine and free rhodamine. Encapsulation innanolipogel increased both the total initial dose to most tissues aswell as the cumulative dose over three days.

Example 5: Cytotoxic and Safety Studies

Materials and Methods

Cell titer blue (Invitrogen) was used as a cell viability markeraccording to the manufacturer's instructions. Chinese Hamster Ovary(CHO) cells (ATCC) were placed in 96 well plates at a density of 5×10⁴cells/well (except the standard, which contained a serial dilution ofthe number of cells). Cells were incubated for 24 hours at 37° C. withserial dilutions of IX PBS (positive control), sodium azide (negativecontrol; Sigma), liposomes, or nanolipogels. Liposomes were fabricatedsimilarly to nanolipogels but, after lyophilization, were reconstitutedwith pure IX PBS and were not subjected to UV irradiation. Nanolipogelswere from the nLG-Empty group. After 24 hours the cell titer bluereagent was added (20 μL/100 volume). The cells were further incubatedfor 4 hours at 37° C., after which they were pelleted and thefluorescence of the supernatant was measured. 100% cell survival isdefined as the average of survival from the IX PBS group and 0% survivalthat from the azide group. All samples were run in triplicate and theexperiment was repeated three times with similar results.

To examine the in vivo safety of nanolipogel particles, C57/Bl6 micewere administered a single intravenous dose of nanolipogels and acutetoxicology was measured 7 days later. Lung toxicity was evaluated byhistology to determine if systemically administered nanolipogels inducedany acute inflammation.

Results

No statistically significant toxic effects were observed from theadministration of empty nanolipogels or nanolipogels co-encapsulatedwith SB505124 (SB) or IL-2. No hepatoxicity was observed, as measured byserum levels of alkaline phosphatase and alanine aminotransferase.Normal physiological reference ranges given by the IDEXX VetTest® systemfor mouse alkaline phosphatase and alanine aminotransferase were 62-209IU/L and 28-132 IU/L, respectively. Furthermore, no renal toxicity wasobserved, as blood urea nitrogen levels were within the normal mousereference range of 18-29 mg/dL. A complete blood count was alsoperformed to identify any hematological toxicity. Leukocyte counts,platelet counts, and hemoglobin content were all within normalphysiological ranges for mouse (leukocytes: 1.8-10.7×10³ cells/uL;platelets: 592-2971×10³ cells/uL; hemoglobin: 11.0-15.1 g/dL).Hematoxylin and eosin staining of lungs demonstrated no obviouspulmonary toxicity. Bronchiolar and alveolar structures appeared normal,and no disruption to epithelial layers or inflammatory infiltrates wereobserved in lung sections.

The in vitro results demonstrate that nanolipogels have similarnegligible toxicities to liposomes.

Healthy C57/Bl6 mice were administered a single intravenous dose ofnanoparticle combination therapy or controls and acute toxicologymeasured 7 days later. No significant toxicities were observed in serummeasurements of alkaline phosphatase or serum alanine aminotransferase.Normal physiological ranges for mouse alkaline phosphatase areapproximately 62-209 IU/L and for alanine aminotransferase approximately28-132 IU/L. No renal toxicity was observed, as blood urea nitrogenlevels were within the normal mouse reference range of 18-29 mg/dL.Complete blood counts demonstrated that normal physiological rangesleukocyte counts, platelet counts, and hemoglobin content. Lung toxicitywas evaluated by histology to determine presence of acute inflammation.Hematoxylin and eosin staining of lungs demonstrated no obviouspulmonary toxicity or inflammatory infiltrates; bronchiolar and alveolarstructures appeared normal with no disruption to epithelial layers.

Example 6: Comparative Distribution of Nanolipogel Carrier andEncapsulant

Materials and Methods

The distribution of both nanolipogel carrier and encapsulated drug payload was investigated using dual-labeled nanolipogels.Fluorescein-labeled phosphoethanolamine was incorporated into the lipidcomponent of rhodamine-loaded nanolipogels. Spectrofluorimetric analysisat 540/625 nm and 490/517 nm demonstrated a dose-dependent fluorescencewith no spectral overlap. There was controlled release of rhodamine, butnot lipid.

Mice bearing subcutaneous B16 tumors received a single IV (tail vein)injection of dual-labeled NLG. Animals were sacrificed at 1, 2, 3, and 7days post injection and tissues collected for homogenization,extraction, and quantification of rhodamine and fluorescein-PE.

Results

Analysis of serum showed prolonged circulation of both encapsulant anddelivery vehicle. Similar patterns of biodistribution were observedbetween lipid and drug payload, with highest accumulations of drugoccurring in the lungs and liver.

Example 7: Lipid Encapsulated Dendrimers for Combined Delivery ofNucleic Acids, Proteins, and Drugs

The nanolipogel encapsulates a dendrimer. The main shell is a liposomeencapsulating a drug and siRNA/dendrimer complex which inserts in cells.Dendritic polymers (dendrimers) are a class of monodisperse polymersdistinguished by their repeated branching structure emanating from acentral core. This branching, which is inherent in the divergentsynthesis of dendrimers, leads to a geometric growth of the polymer thatcan nearly approximate a sphere with increased branchings or highergenerations (generation 6 or above). This branching creates a coreideally suited for entrapment of a variety of small hydrophobicmolecules such as drugs as well as complexation of nucleic acids. Forexample, Superfect®, a commercially available activated dendrimertransfection agent. Combined with their narrow molecular weightdistribution and small size (less than 10 nm), dendrimers have beenutilized for a large number of applications including drug and genedelivery. Dendrimers complexed with nucleic acids can be cleared rapidlyupon in vivo administration and hence protective targeting of thiscomplex would be a more attractive modality for site-specific delivery.The liposomal formulation serves two functions: 1) Protectiveencapsulation of siRNA complexed liposomes and 2) facilitating deliveryof small molecule hydrophilic drugs such as rivoavrin or proteins suchas IFNa. Complexation of the inner dendrimer core with siRNA: Nucleicacids are generally stabilized by cationic polymers in a polyplexformation akin to the physiological packaging of nucleic acids aroundhistones. Cationic polyamidoamine (PAMAM) dendrimers, generation 5 (G5)5.4 nm, serves this purpose.

Materials and Methods

siRNA/Dendrimer polyplexes are formed by combining G5 PAMAM and siRNA atan amine to phosphate (N/P) ratio of 1:1 to 10:1. The precise ratiowhich will yield optimal silencing can be determined by mixing stocksiRNA and PAMAM at different molar ratios for 30 min at room temperaturein sterile 10 mM HEPES buffer, pH 7.2 with light vortexing. Thisprocedure yields a siRNA-dendrimer polyplex with a charge (zetapotential) of +20 or above and effective diameter of 10 nm, which issuitable for encapsulation in liposomes. Next, the polyplex isco-encapsulated with the drug (IFNa and/or Ribavirin) in the liposomalparticle. A dehydrated lipid film comprised ofdistearoyl-glycero-phosphocholine (DSPC), cholesterol, anddistearoylglycero-phosphoethanolamine (DSPE) with an amine terminatedpolyethylene glycol (PEG2000) spacer (DSPE-PEG2000-NH2) is first mixedin the molar ratio of 65:30:5, then rehydrated under sonication with a10 mg/ml solution of siRNA/Dendrimer polyplex and drug. The ratio ofdrug to siRNA/dendrimer in solution can be tuned during formulation.Optimal ratio is dictated by in vitro and in vivo efficacy studies. Theintrinsic “built-in” lipid PEGylation facilitates a longer circulationtime compared to particles without PEG. PEG incorporation yields asteric hydration barrier shield which facilitates long-lived in vivocirculation, (i.e avoidance of the reticuloendothelial system andnon-specific uptake by macrophages).

Following the mixing of drug and siRNA/Dendrimer polyplex in presence oflipids, the solution is extruded through a series of filters. Firstthree times through a 5 μm filter, three times through 1 μm filter, andfive times through a 200 nm filter collecting extrudate in a steriletube. Excess siRNA complex, drug and lipids are removed by spinning for45 minutes at 24000 rpm at 4 C (3×) in an ultracentrifuge.

FIG. 4A is a schematic of LED preparation encapsulating siRNA/dendrimerpolyplex and drug combinations, with covalent modification of the outershell with targeting antibodies or single chain variable fragments(scFv): Attachment of antibodies or scFv to the amine terminatedliposome is achieved by activating the protein in 0.1 MES buffer (pH5.5) in the presence of ethyldicarbodiimide and N-hydroxysuccinimide for10 min followed by addition to particles in buffered saline (pH 7.4).This reaction activates carboxylate groups on the protein for covalentlinkage to exposed amine groups on the particles. Initially, thereaction stoichiometry is adjusted to yield an approximate density of1-10 scFv molecules per particle, however, this density can be easilyincreased by varying the stoichiometry of the reaction to facilitatemaximal internalization. Total time for the reaction is 30 min at roomtemperature. These reaction conditions have no effect on the integrityor function of encapsulated agents.

Results

LEDs can facilitate drug internalization as depicted in FIG. 4A withmacrophages in culture. The drug Methotrexate (MTX) was used as a modeldrug. FIG. 4B shows the cytotoxicity of LED and LED encapsulating themodel drug methotrexate (MTX). Bars indicate successive dilutions of LEDor drug or combinations starting from (1 mg/ml left to right to 10ug/ml). Azide is used as a positive control for cell killing. Startingat 10%, left to right, and increasing to 1%. Compared to free drug(MTX), LED containing MTX were slightly less toxic, presumably becauseof drug sequestration. LEDs alone showed no cytotoxicity. LEDsencapsulating the dye rhodamine facilitate internalization andcytoplasmic localization of the dye and LEDs containing the the pGFPplasmid showed enhanced efficiency in transfection of macrophagescompared to a standard transfection agent such as lipofectamine.

LEDs encapsulating the dye rhodamine facilitate internalization andcytoplasmic localization of the dye and LEDs containing the pGFP plasmidshowed enhanced efficiency in transfection of macrophages compared to astandard transfection agent such as LIPOFECTAMINE®.

To determine if terminal amine groups on PAMAM dendrimers provideendosomal buffering and disrupt endosomes by the proton sponge effect,an Acridine Orange (a dye whose spectral properties change depending onits location in endosomes or cytosol) assay was used with BMDCs, whichwere treated with unmodified generation 4 PAMAM dendrimers (G4), ordendrimers conjugated to cyclodextrin molecules (CD) that substitutedand shielded primary amines with or without ionophorecarbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). The resultsindicate that of the tested combinations, unmodified G4 dendrimer wasbest at endosomal disruption followed by G4-3CD (FIG. 4C). G4-6CD wasthe least effective at endosomal disruption of the combinations tested,supporting the idea that proton sponge effect is mediated by primaryamines and substituting amines with CD decreases buffering capabilities.

LEDs were also tested using different dendrimer (G)-cyclodextrinconjugates (CDs) and Nitrogen/Phosphorus (N/P) ratio, and compared tovector delivery (pGFP) using LIPOFECTAMINE® 2000 and liposomes in avariety of cells types. CD significantly affected dendriplex (FIG. 4D).Dendriplexes (from modified dendrimers) transfect better thanLipofectamine 2000 in BMDCs. LEDs also transfected better than liposomesencapsulating vector in BMDCs.

LEDs encapsulating siRNA against CD4 or Luciferase (Luc) and surfacefunctionalized with anti-CD7 were test for the ability to mediateinternalization target mRNA knockdown in Jurkat (human T cell line)cells. Results indicated that LED delivered siRNA reduced surfaceexpression of CD4, or Luc relative to controls (FIG. 4E).

In a second experiment, LEDs including 200 ug of dendrimer and 400 pmolsiGFP. (Ctl=LFA:siGFP) were utilized to knockdown GFP expression is astably transfected cell line. Stable 293T-eGFP cells were treated withdendrimers for 4 h in SFDMEM followed by examination of GFP expression.Cells treated with most dendrimer (G)-cyclodextrin conjugates (CDs)combinations exhibited greater reduction in GFP expression compared tomock and LIPOFECTAMINE®: siGFP controls (FIG. 4F).

Example 8: Antigen Cross Presentation with Lipid Encapsulated Dendrimers

Materials and Methods

Mouse Bone Marrow-Derived Dendritic Cells (BMDCs) were incubated withliposomes encapsulating ovalbumin (OVA) alone, dendrimer alone, or bothOVA and dendrimer (LED).

Results

Controls of cells and empty liposomes showed undetectable levels of 25.D16 antibody staining, which binds MHC Class I-SIINFEKL complexes. Cellsreceiving LEDs showed the highest level of antigen cross-presentation.(See FIG. 5A). * p<0.05 by one-way ANOVA Bonferroni post-test.

Example 9: Vaccine Delivery with Lipid Encapsulated Dendrimers

Materials and Methods

Antigen Presentation

1×10⁵ BMDC/well (96 well plate)+25 uL liposomal particles. Particlegroups:

a. −/− (nothing outside nothing inside particles)

b. −/OVA (nothing outside, OVA encapsulated)

c. −/G5+OVA (nothing outside, OVA and G5 dendrimer inside)

d. −/G5+OVA+CpG

e. MPLA/− (MPLA outside, nothing inside)

f. MPLA/OVA

g. MPLA/OVA+G5

h. MPLA/OVA+G5+CpG (MPLA outside; OVA, G5 dendrimer, CpG encapsulated)

Where OVA=ovalbumin, MPLA=monophosphoryl lipid A, G5=generation 5dendrimer, CpG=CpG oligonucleotide (TLR9 ligand). Treatment wasincubated with BMDC for 24 hours followed by 4 day co-incubation with WTsplenocyte.

Analysis of pro-inflammatory cytokine production BMDCs were incubatedwith liposomal nanoparticles encapsulating antigen andsurface-functionalized with increasing amounts of TLR ligand CpG for 24hours before supernatant analysis by ELISA.

Results

Cells were stained with 25.D16-PE, an antibody that is specific formouse MHC Class I-SIINFEKL complexes, as assessed for antigencross-presentation by flow cytometery. The results indicated that −/OVAparticles induce some cross-presentation, which was increased by OVAparticles containing dendrimer. Particles the combination of MPLA, CpG,and dendrimer induce the highest amount of cross-presentation. (See FIG.5B). Liposomal nanoparticles surface functionalized with CpG alsoinduced a dose-dependent increase the production of pro-inflammatorycytokine IL-6. (See FIG. 5C).

Example 10: Preparation and Characterization of Nanolipogels ContainingMycophenolic Acid (MPA)

A system consisting of a biodegradable, cyclodextrin-based hydrogelencapsulated within a lipid bilayer, was used to deliver molecules thatselectively immunosuppress an individual with an autoimmune disease.

Materials and Methods

Fabrication of Lipogel Nanoparticles

Hydrogel-based nanoparticles (nanolipogels) were fabricated by remotelyloading liposomes with a diacrylate-terminated co-block polymer ofpoly(lactic acid) and poly(ethylene glycol) in the presence of aphotoinitiator (FIGS. 6B and 6C). Mycophenolic acid (MPA), which wascomplexed with a cyclodextrin derivative to improve its aqueoussolubility, was also remotely loaded into the liposome. This loadingprocess involved rehydrating lyophilized, preformed liposomes in aqueoussolution with these precursors and drug. Photopolymerization of thesenanoparticles under UV light induced cross-linkage between theacrylated-precursors and gelation of the particle interior into a stablematrix. These nanolipogels had an average hydrodynamic diameter of 225nm, with a median diameter of 203 nm and mode diameter of approximately141 nm. See FIG. 7A.

To prepare liposomes, a mixture of 2:1:0.1 molar ratio ofphosphatidylcholine:cholesterol:phosphatidylethanolamine (Avanti Polar)in chloroform was evaporated under nitrogen gas stream and thenlyophilized. The resulting dry lipid film was then rehydrated with PBSto form large lipid vesicles. This large lipid vesicle suspension wasextruded five times through a 200 nm pore filter (Whatman) and then fivemore times through a 100 nm pore filter. Lipid nanoparticles were thensurface modified with anti-CD4 targeting antibodies (clone RM4-4, BDBiosciences) using sulfo-NHS/EDC (Pierce) covalent conjugation, and thenlyophilized overnight.

Next, 20 mg of mycophenolic acid (Sigma) in 400 of methanol wascomplexed to 100 mg of a mixture of aminomethacrylate (Sigma) andsuccinylated β-cyclodextrin (CTD Holdings) and 300 mg of hydroxypropylcyclodextrin (CTD Holdings) in PBS under vigorous mixing for 15 min atroom temperature. The methanol was evaporated from the MPA-cyclodextrinmixture, and the remaining cyclodextrin-MPA mixture was resuspended withIrgacure 2959 (Ciba) photoinitiator and a PEG-oligomer consisting of4000 Da linear poly(ethylene glycol) flanked on each side byapproximately 1-2 lactic acid monomers with a terminating acrylategroup. The synthesis of this crosslinkable oligomer is described bySawhney (Sawhney, et al., Macromolecules, 26:581-587 (1993)).

Lyophilized liposomes were then remotely loaded with the aqueousMPA-cyclodextrin-Irgacure-PEG mixture. Vigorous mixing was applied for30 min. The liposomes were then cross-linked under a 430 W UV lamp withUVA light (315-400 nm transmission filter) for 8 min on ice to form thenanolipogel, then rinsed in PBS and collected by ultracentrifugation at98,205 rcf for 1 hr at 4° C. Nanolipogels were stored at −20° C. untiluse.

PLGA nanoparticles encapsulated with MPA were synthesized andcharacterized as previously described (Shirali, et al., Am J Transplant,11(12):2582-92 (2011).

Nanoparticle Characterization

The hydrodynamic diameter of nanoparticles in PBS was measured with anNS500 nanoparticle tracking system (Nanosight, Amesbury Wilshire, UnitedKingdom). For transmission electron microscopy analysis, nanolipogelsamples were stained with osmium tetroxide and then imaged on an FEITenai Biotwin microscope. Scanning electron microscopy was performed ongold-sputtered PLGA nanoparticles, and imaged with an XL-30 ESEM-FEG(FEI, Hillsboro, Oreg.) microscope. The amount of MPA loaded innanoparticles was determined from the supernatant of nanoparticlesdissolved in 1% triton X-100 in 0.1 M NaOH for at least 1 hr at 37° C.;the fluorescence of MPA was measured using an excitation wavelength of340 nm and an emission wavelength of 450 nm on a SpectraMax plate reader(Molecular Devices).

Results

In Vitro Characterization of a Hydrogel-Based Nanoparticle

These nanolipogels had an average hydrodynamic diameter of 225 nm, witha median diameter of 203 nm and mode diameter of approximately 141 nm.Gelation of the nanoparticle interior into a stable matrix wasdemonstrated by exposing these nanolipogels to surfactant; thephoto-cross-linked matrices retained the diameter of the particlewhereas conventional liposomes ruptured upon surfactant treatment (FIG.7D). The representation of these nanolipogels as hydrogel particulateswith a lipid exterior was confirmed by transmission electron microscopy,which showed that lipid-specific osmium tetroxide staining ofcryosectioned samples had a localized staining pattern confined to theexterior membrane of the particle. These nanolipogels could be loadedwith bioactive mycophenolic acid and sustain its release. MPA that wasloaded maintained its potency and was released in a sustained fashionfor over a week. The average MPA loading in these nanolipogels wasapproximately 6.65±3.82 μg MPA/mg particle, with an encapsulationefficiency of approximately 3.79% (FIGS. 8A and 8B). In contrast,liposomes with the same lipid composition as the nanolipogelsencapsulated less drug, with a loading of 2.03±0.14 μg MPA/mg particle(FIG. 8A), and an encapsulation efficiency of 1.37% (FIG. 8B).Consistent with the swelling nature of hydrogel nanoparticles, a modestincrease in the effective hydrodynamic diameter of the particles wasobserved following photopolymerization (FIG. 8C). The formation of astable interior was verified by exposing nanogels to triton X-100:liposomes ruptured upon exposure to this surfactant, whereas lipogels donot. PBS is phosphate buffered saline at pH 7.4 (FIG. 8D). Singleparticle counting verified that triton X-100 ruptured and decreased thenumber of liposomes but not nanogels (FIG. 8E). Horizontal dashed lineindicates the limit of detection of the Nanosight particle trackinginstrument (FIG. 8E). In FIG. 8C-8E, error bars represent standard errormeasurement, with at least 3 measurements per group. * p<0.05 or less by1-way ANOVA with Bonferroni multiple comparison test.

Example 11: Functionalization of Nanolipogels and Use of FunctionalizedNanolipogels to Target CD4 T Cells

Materials and Methods

To achieve CD4 T cell targeting potential with these particles, anon-depleting antibody for CD4 was covalently attached to the surface ofthe particle. To test the cell-specific binding capacity of the CD4targeted particles, fluorescently labeled particles were incubated withsplenocytes ex vivo, and the colocalization of particle fluorescencewith leukocyte specific subsets determined via flow cytometry. CD4 Tcells could specifically attach to CD4 targeted nanoparticles.

In order to demonstrate that these CD4-targeted, MPA-loadednanoparticles could inhibit CD4 T cell proliferation, the proliferativecapacity of CD4 T cells in vitro was measured, after exposing the cellswith CD4-targeted and non-targeted MPA-loaded particles. CD4 T cellswere briefly incubated with particles, washed to remove any unboundparticles, and then stimulated for 4 days with anti-CD3 and anti-CD28antibodies to promote proliferation.

Results

Experiments were conducted to determine whether nanolipogel particlescould be functionalized and used to target CD4 T cells and suppresstheir proliferation. In lupus, CD4 T cells provide costimulatory signalsto B cells which leads to the production of pathogenic autoantibodies.Depletion of CD4 T cells has been shown to provide therapeutic benefitin murine models of lupus (Wofsy, et al., J Exp Med, 161(2):378-91(1985); Wofsy, et al., J Immunol, 138(10):3247-53 (1987)).

In incorporate this strategy, CD4 antibody (clone RM4-4) were covalentlyattached to nanolipogels. CD4 T cells treated with CD4-targeted, MPAloaded nanoparticles had impaired proliferation and cytokine productionwhereas CD4 T cells that were treated with non-targeted nanoparticles orfree MPA (drug not encapsulated in particles) had normal proliferationand cytokine production (FIG. 8F). These results indicate that only theCD4-targeted nanoparticles could remain bound to the CD4 T cells, evenafter a wash step, and that the encapsulated MPA in these boundsparticles was subsequently released into the cells to inhibit theirproliferation.

Example 12: Toxicology Studies in Mice

Materials and Methods

Toxicology Studies

Acute toxicity studies were performed in 10 week old C57BL/6 femalemice. Mice were dosed with indicated treatment groups on day 0, 1, 2,and 3. After four daily doses of 0.625 mpk MPA in particles wereadministered, complete blood counts and clinical chemistries for liverand kidney function were measured on 4, 7, and 14 days after the firstdose. Serum concentrations of alkaline phosphatase (ALKP), alaninetransferase (ALT), total bilirubin (tBIL), and blood urea nitrogen (BUN)were measured using reagents from Teco Diagnostics.

Blood was collected in EDTA spray-coated tubes, and immediately analyzedby complete blood count (CBC) on a Hemavet blood counter. Urine wascollected by bladder compression, and analyzed with Uristix test(Bayer).

Renal Analysis and Histology

Urinalysis was performed with Uristix assays. Proteinuria measurementscorresponding to 300 mg/dL or greater were considered a positive test.Leukocyte esterase content in the urine that corresponded with 10 ormore leukocytes{circumflex over ( )}L was considered a positive test.Renal function was assessed by the blood urea nitrogen (BUN) content inserum, using a kinetic assay (Teco Diagnostics).

For histology analysis, kidneys from 36-40-weeks-old mice were fixed in10% neutral buffered formalin, and hematoxylin and eosin stainedsections were prepared by the Yale University Pathology HistologyService. Tissues were imaged on a Nikon TE-2000U microscope with a NikonDS Fil color camera and NIS Elements AR software (version 2.30). Scoringof glomerular damage and intertubular infiltration was performed by M.K.

Results

The dosing regimen used with MPA-loaded nanolipogels did not induce anyapparent systemic toxicity, as measured by body mass (FIG. 9A), renaland liver functional enzyme tests (alkaline phosphatase, FIG. 9B;Alanine transferase, FIG. 9C; blood urea nitrogen, FIG. 9D, and totalbilirubin (FIG. 9E) and complete blood count analysis (leukocytes, FIG.10A; platelets, FIG. 10B; hemoglobin, FIG. 10C; hematocrit, FIG. 10D).White blood cell, platelet, hemoglobin, and hematocrit levels werewithin normal physiological ranges (FIG. 10A-D). Furthermore, no liveror renal toxicities were observed. Body weight and serum concentrationsfor alkaline phosphatase, alanine transferase, total bilirubin, andblood urea nitrogen were normal (FIG. 10A-E).

A complete blood count (CBC) suggested that the particulate-mediatedimmunosuppression is not accomplished by blunt reduction in total whiteblood cell numbers, as the total white blood cell numbers are normal andnot reduced after particulate therapy. This observation indicatesimmunosuppression would be observed in the activation state of immunecell subpopulations.

This data shows that MPA-loaded nanolipogels did not induce any apparentsystemic toxicity. This improved safety with nanoparticle therapy standsin contrast to the side-effects often observed with conventionaladministration of immunosuppressants (i.e. drug not encapsulated withinnanoparticles), which often leads to toxicity in the bone marrow(myelotoxicity), cytopenias, anemia, or organ damage.

Example 13: The Effect of MPA-Loaded Nanolipogels on Survival ofLupus-Prone NZB/W F1 Mice

Materials and Methods

Statistical Analysis

Statistical analyses were performed in GraphPad Prism (version 5.03).For comparisons in survival studies, the log-rank (Mantel-Cox) test wasused. Experimental comparisons with multiple groups used AN OVA analysiswith Bonferroni post-test. Two-tailed t-tests were used for somecomparisons, as indicated in figure captions. A p-value of 0.05 or lesswas considered statistically significant.

Animal Studies

The in vivo therapeutic efficacy of nanolipogel particles was tested infemale NZB/W F1 mice, a lupus-prone animal model. NZB/W F1 mice developauto immunity that resembles human lupus nephritis, with development ofglomerulonephritis, anemia, and the generation of autoantibodiesspecific for double-stranded DNA and other self-antigens. NZB/W F1 micewere administered a life-long, single weekly intraperitoneal injectionof nanoparticle therapy beginning at 18 weeks of age, before the onsetof proteinuria. The single, weekly MPA dosage in nanoparticles was 0.625mg of MP A per kg animal (mpk). Control mice received only salinebuffer.

Animal studies using female NZB/W F1 mice (The Jackson Laboratory, stock#100008) were performed with the approval of the Yale UniversityInstitutional Animal Care and Use Committee. Beginning at 18 weeks ofage, NZB/W F1 mice were administered a weekly intraperitoneal injectionof nanoparticles or mycophenolic acid in buffer for the entirety oftheir life. An intraperitoneal injection was used, as opposed tointravenous tail vein injection, to prevent long-term tail vein damagefrom chronic injections. Mice urine was collected by bladdercompression, and blood was collected via retroorbital bleed withisoflurane anesthesia. Mouse survival was continued until symptoms ofcachexia (more than 5-10% body weight loss), lethargy, or poor conditionwarranted humane euthanization.

Nephritis occurs upon autoantibody immune complex deposition within thekidneys and subsequent infiltration by T cells, neutrophils, andmacrophages. This inflammatory damage was monitored longitudinally bythe presence of elevated levels of proteinuria and leukocyte esteraseactivity in the urine.

The efficacy of particulate therapy after the onset of establishedproteinuria (more than 2 consecutive daily readings of >300 mg/dLprotein in urine) was also evaluated.

Results

Treatment with nanoparticle therapy had a statistically significanteffect on extending the median survival age of mice by 3 months. NZB/WF1 mice that received CD4 targeted particles loaded with mycophenolicacid had a median survival time of 50 weeks compared to only 38 weeksfor mice receiving no therapeutic immunosuppression. (FIGS. 11A and 11B,14B). The delivery of mycophenolic acid in particles was critical toachieving therapeutic benefit. The administration of an equivalent MPAdosage given soluble in buffer (referred to as “free MPA”) at 0.625 mpkhad no effect on extending survival (FIG. 11C). When the free MPA dosagewas increased by 16-fold, to 10 mpk, there still was no improvement insurvival (FIG. 11C).

The data shows that all treatment groups that received MPA-loadednanoparticles had extended median survival times. CD4-targetedMPA-loaded particles extended survival to approximately 50 weeks(p<0.0083), and non-targeted MPA-loaded particles extended survival toapproximately 51 (P<0.0304) (FIGS. 11A-11C). Thus nanoparticles uniquelyenhance the immunosuppression of MPA, compared to what is typicallyachieved with much higher and more frequent doses of free MPA (30 to 100mg/kg/day) (Ramos, et al., Nephrol Dial Transplant, 2003: 18(5):878-83(2003); Lui, et al., Lupus, 1 1(7):411-8 (2002)) and thatnanoparticulate therapy was not dependent on direct targeting ofnanoparticles to CD4 T cells. The in vivo use of nanoparticles wassafe—no acute toxicity was observed when tested in C57BL/6 wild typemice, and no chronic toxicity was obvious in NZB/W F1 mice during theyear-long therapeutic studies.

Nanogels also protected against nephritis. The extension in survivalwith particulate therapy correlated with a delay in nephritis, which isthe primary cause of mortality in NZB/W F1 mice. Mice receivingprophylactic treatment particulate therapy had delayed onset ofproteinuria (FIGS. 12A and 12B) and presence of leukocyte esterase inthe urine (FIGS. 12C and 12D) compared to free drug and buffertreatments. Renal function, as measured by blood urea nitrogen (BUN)levels, was also preserved in particulate treated mice (FIG. 12E). Thepercentage of mice with abnormally elevated blood urea nitrogen (BUN)levels (greater than 18-29 mg/dL), which is an indicator of impairedrenal function, was lower in 36-40-weeks-old mice Consistent with theseobservations, histopathologic evaluation of kidneys from mice atapproximately 36-40 weeks of age verified that particulate-treatedgroups had decreased kidney damage and inflammation (FIGS. 12F and 12G).Treatment extended survival (FIG. 12H).

Mice that began weekly particulate therapy after the onset ofproteinuria also had extended survival (FIG. 12I). Survival time lasted12 weeks with particles compared to only 4 weeks with buffer (p<0.0198)when administered to mice after the onset of proteinuria, indicatingthat the particulate therapy is still efficacious even under morestringent disease conditions.

Example 14: Nanolipogel Particle Biodistribution Studies

The data discussed above shows that a CD4 directed nanoparticle therapyis successful in preventing T-dependent autoimmunity by exclusivelyabrogating CD4 T cell activation or proliferation. However,nanoparticles without any CD4 targeting also provided therapeuticbenefit in vivo. To investigate a basis for this therapeutic benefitwithout CD4 targeting, the in vivo biodistribution of nanoparticles wasanalyzed.

Materials and Methods

Particle Biodistribution Studies

Rhodamine loaded nanolipogels were prepared and then injectedintraperitoneally into mice. For organ biodistribution and histologystudies, 12-week-old NZB/W F1 mice were used. Organs were harvested,weighed, and imaged with the IVIS imaging system to obtain quantitativerhodamine fluorescence measurements. For histological analysis, spleenswere snap-frozen in OCT embedding medium and then sectioned on acryotome onto charged slides. Sections were fixed in ice cold acetonefor 10 minutes, and subsequently stained with antibodies for CD4, CD19,or F4/80. Tissue sections were imaged on a Nikon TE-2000 microscope.FITC channel: 470/40, 525/50; rhodamine channel: 540/25, 605/55;Alexa647 channel: 620/60, 700/75.

The extent to which different lymphocyte and antigen presenting cellsubsets were associated with nanoparticles was also investigated.

Flow Cytometer Analysis

For flow cytometric measurement of particle binding to immune cells, theanalysis was performed in C57BL/6 mice immunized with 1×10⁶ sheep redblood cells (Colorado Serum Company). Seven days after immunization,rhodamine-labeled particles were injected, and 2-3 hr later, cells fromthe spleen, lymph nodes (pooled inguinal, brachial, and cervical), andbone marrow were harvested and then analyzed the same day on an LSR IIflow cytometer. Peripheral blood lymphocytes were collected in asolution of 50 U/mL heparin and isolated with Ficoll gradientseparation. Splenocytes were harvested and red blood cells were removedwith ACK lysis buffer.

Lymphocytes were blocked with 1% fetal bovine serum (FBS) in PBS, andthen stained with the following combinations: for T cells—CD4 (cloneRM4-5), CD8, CD62L, CD44, PD-1, and CXCR5-biotin; for B cells—B220,CD138, IgD, GL-7, PNA-biotin (Vector Labs), and MHC II; and for innateantigen presenting cells-F4/80, CD11c, PDCA-1, CD40, CD80, and MHC II(all antibodies from BD Biosciences or eBioscience). Streptavidin Pe-Cy7(Invitrogen) was used as a detection agent for biotinylated antibodies.

Cells were fixed with 2% paraformaldehyde and then analyzed on an LSRIIflow cytometer (BD Biosciences). For in vitro proliferation studies, Tcells were labeled with 0.5 μM CFDA-SE (Invitrogen) in PBS for 10 min at37° C. at a concentration of 4×10⁶ cells/mL, and then washed twice withRPMI-1640 complete media (10 mM HEPES, 1 mM L-glutamine, 100 U/mLpenicillin, 100 μg/mL streptomycin, 50 μM β-mercaptoethanol), with 10%heat inactivated FBS. For intracellular IFN-γ staining, 1×10⁶splenocytes were first stimulated with 20 ng/mL PMA and 2 μg/mLionomycin for 3 hr in brefeldin A (BD Bioscience) at 37° C. in completeRPMI-1640 media. Cells were labeled for extracellular CD4 and CD25,treated with fixation/permeabilization buffer (eBioscience), and thenstained with antibodies for Foxp3 and IFN-γ according to manufacturer'sspecifications. Cells analyzed for intracellular IFN-γ expression wereprestimulated with PMA and ionomycin for 3 hr in brefeldin A (BDBioscience) before staining.

Results

Flow cytometer analysis demonstrated that CD4 targeted nanoparticlesassociate with CD4 T cells in the spleen. Notably, splenic macrophagesand both conventional and plasmacytoid dendritic cells also internalizednanoparticles (FIG. 13A). Histological examination of spleens showedthat nanoparticles could localize to the splenic T cell zone and redpulp after 2-3 hours. T and B220⁺ cells associated with particles, withthe greatest binding (based on the percentage of the cell subset)observed with dendritic cells, macrophages, and plasmablasts (FIGS. 13Aand 13B). Furthermore, nanoparticles also trafficked to the bone marrow,and could be internalized by bone marrow resident plasmacytoid dendriticcells and CD11b macrophages (FIG. 13C).

Nanolipogels Target Immune Cell Subsets In Vivo

Regardless of targeting, nanogels had greater accumulation in thespleen, kidneys, liver, heart, lung, and pancreas compared to freerhodamine (FIG. 13D-13J). This trend is consistent with other reportsthat show particulate drug delivery systems can increase thebioavailability of compounds (Hillery, et al., Drug delivery andtargeting for pharmacist and pharmaceutical scientists, New York City,N.Y., Taylor and Francis (2001)). Analysis of fluorescently labeledparticles injected into mice showed that nanoparticles, even without CD4targeting capability, persisted longer in tissue compared to dyeadministered soluble in buffer (spleen, 13D; heart 13E; lung, 13F;kidney, 13G; liver, 13H; pancreas, 13I). This enhanced distribution timeof particles and their encapsulants in tissue is consistent with reportsthat demonstrate that nanoparticle drug delivery systems can betterprolong the bioavailability of compounds.

Example 15: In Vivo Immune Responses to Particulate Therapy

To further characterize the results obtained with nanoparticulatetherapy, the immune responses in all treatment groups was analyzed.

Materials and Methods

Antibody ELISAs

As an initial proxy for evaluating these cellular responses, the levelsof anti-double stranded (ds) DNA antibodies in the serum were monitoredas a potential explanation for particulate-mediated protection againstnephritis. Autoantibodies promote pathology, and many reports have shownthat amelioration of autoantibody titers can lead to protection fromnephritis. For dsDNA ELISAs, a coating layer of 10 μg/mL methylated BSA(Calbiochem) in bicarbonate buffer was adsorbed onto a high-binding96-well plate, followed by incubation with 10 μg/mL of calf thymus DNA(Sigma). 1% BSA was used as a blocking agent and diluent, and 0.05%TWEEN®-20 in PBS was used as a wash buffer. Horseradishperoxidase-conjugated antibodies for mouse-specific pan-IgG (ICL Labs),IgG1, IgG2a, IgG2b, and IgG3 (Invitrogen) were used for detection,followed by TMB (KPL) substrate development.

Results

White blood cell counts were not reduced after 4 consecutive, dailydoses of nanogels in wild type mice, and surprisingly, at the dosageused with the nanoparticle therapy, there was only marginal inhibitionin the development of anti-ds DNA antibodies (FIG. 14A-14E). At 36-40weeks of age, no differences were observed in the percentage of splenicgerminal center B cells or CD4 T follicular helper (FIG. 15A-15B).Furthermore, there were only mild reductions in the percentages ofperipheral blood CD138^(hi)B220^(lo) antibody-secreting B cell (FIG.15C). These results indicate that the reduction in autoantibodyresponses was not the primary consequence of nanoparticle therapy.

Inflammatory responses by CD4 T cells are known to worsen disease; forexample, Th1 responses are often elevated in both human patients andmouse models of lupus. The data shows that percentage of splenic IFN-γproducing CD4 T cells was statistically significantly reduced byapproximately 2-fold, from 10% of CD4 T cells (in untreated controls) to5% of CD4 T cells (FIG. 16A). This correlated with mild reductions inthe percentage of activated splenic and peripheral blood CD4 T cells(CD62L^(lo)CD44^(hi)) (FIGS. 16B and 16B). This reduction ininflammatory CD4 T cell responses did not correlate with any expansionin CD4 T regulatory (T reg) cell numbers (FIG. 16D). The percentage ofsplenic IFN-γ producing CD4 T cells was significantly reduced byapproximately 2-fold (FIG. 16C), from 10% of CD4 T cells in untreatedcontrols to 5% with particle treatment. Collectively, these dataindicate MPA-loaded nanolipgel therapy modulates T cell phenotypewithout causing lymphopenia, and that immunosuppression occurs throughmodulating immune cell function or activation state rather than bylymphodepletion.

Conventional dendritic cells (cDC) also had mild reductions inexpression of stimulatory markers such as CD40 and MHC II (FIGS. 17A and17B), respectively). This trend was not observed in macrophages (FIGS.17C and 17D) or plasmacytoid dendritic cells (FIGS. 17E and 17F).Particulate-mediated reduction in stimulatory activity in cDCs maycontribute to some of the therapeutic benefit observed. Consistent withthe observations, prior reports indicate the cDCs may contribute tolupus pathology by stimulating T cell activation, but remain dispensableto early T cell-B cell interactions that result in autoantibodyformation (Teichmann, et al., Immunity, 33(6):967-78 (2010).

Example 16: Nanolipogel Immunosuppression Affects Dendritic CellPopulations

Dendritic cells can contribute to lupus disease pathology, and their invivo depletion or pharmacological induction to a less inflammatory oreven tolerogenic state may alleviate lupus autoimmunity. Because antigenpresenting cells such as dendritic cells can internalize nanoparticles,the effect of MPA-loaded nanoparticles on dendritic cell maturation andactivation was explored.

Materials and Methods

Bone marrow derived dendritic cells were treated with MPA-loadednanoparticles and dendritic cell surface marker expression afterstimulation with LPS, was measured. The effect of nanoparticleinternalization plasmacytoid dendritic cells (pDC) inflammatory cytokineproduction was also investigated.

Bone Marrow Derived Dendritic Cell Culture and Mixed Lymphocyte ReactionStudies

Bone marrow cells were isolated from the femurs and tibia of Balb/cmice, and then differentiated into CD11c⁺ dendritic cells using 10 ng/mLGM-CSF (eBioscience) and 5 ng/mL IL-4 (eBioscience) in RPMI-1640complete media (10 mM HEPES, 1 mM L-glutamine, 100 U/mL penicillin, 100U/mL streptomycin, 50 μM β-mercaptoethanol), with 10% heat inactivatedfetal bovine serum. These bone marrow dendritic cells (BMDCs) were dosedwith nanoparticle therapy (125 ng/ml MP A), beginning at day 1 ofculture. Media was changed thereafter every two days with freshMPA-loaded particles in the media. On day 6 of the culture, BMDCs werereplated at 200,000 cells/well in a 96-well round bottom plate, and thenchallenged with 50 ng/mL lipopolysaccharide (LPS) (Sigma) for 18 hr toinduce further maturation. The BMDC purity after 7 days in culture wasconfirmed to be approximately 70% CD11c⁺. For mixed lymphocytereactions, day 7 BMDCs were irradiated with 3000 rad in an X-Rad 320(Precision X-ray) and then co-cultured for 4 days in complete RPMI-1640complete media with purified CD4 T cells from C57BL/6 mice at a ratio of1×10⁵ BMDCs to 2×10⁵ CD4 T cells.

Samples for confocal imaging were prepared by seeding 7-days-old BMDCsonto ProbeOn Plus glass slides (Fisher), incubating withlissamine-rhodamine-labeled particles for 1 hr, and then staining cellswith Alexa 488-phalloidin and TO-PRO-3 (Invitrogen). Cells were imagedwith an LSM 510 Meta confocal microscope (Carl Zeiss)

For plasmacytoid dendritic cell studies, bone marrow cells wereincubated with particles or free MP A for 30 min at 37° C. in RPMI-1640complete media, washed, and then challenged with 1 μM CpG-Aoligonucleotide (ODN 1585 from Invivogen) for 18 hr.

Cytokine Analysis

ELISAs for interleukin-10 (IL-10), interleukin 12p (IL-12p70), tumornecrosis factor α (TNF-α) (eBiosciences) and interleukin 2 (IL-2),interleukin 6 (IL-6), and interferon γ (IFN-γ) (BD Biosciences) wereperformed according to manufacturer's protocols. ELISAs for IL-I β andIFN-α were performed as previously described in (Demento, et al.,Vaccine, 27(23):3013-21 (2009); Lund, et al., J Exp Med, 198(3):513-20(2003), respectively.

Results

Bone marrow derived dendritic cells treated with nanoparticles haddecreased surface expression of the costimulatory markers CD40 (FIG.18A), CD80 (FIG. 18B), and CD86 (FIG. 18C) as well as decreasedexpression of MHC class I and MHC class II molecules (FIGS. 18G and18H). These dendritic cells also had decreased production of theinflammatory cytokines IFN-γ (FIG. 18D), IL-12p70 (FIG. 18E), and TNFa(FIG. 18F). The down-regulation of these surface markers andinflammatory cytokines may thus limit the ability of DCs to activate Tcells. When MPA treated BMDCs were co-cultured with allogeneic CD4 Tcells in a mixed lymphocyte reaction, the CD4 T cells had attenuatedproliferation. Furthermore, these nanoparticle-treated dendritic cellscould promote the partial expansion of CD25 Foxp3⁺ CD4 T cells in vitro.Furthermore, CD11c+ dendritic cells isolated from wild type mice thatwere treated with nanogels elicited weaker production of IFN-γ fromallogeneic CD4 T cells (FIG. 19A).

The suppression of dendritic cell activation markers by MPA isreversible in vitro, and requires the continued presence of MPA in mediato maintain attenuated responses (Mehling, et al., J. Immunol,17:351-363 (2005)). Hence, the slight size of reduction in CD40 and MHCclass II in vivo may be a consequence of the intermittence of weeklydosing. Much greater suppression was observed with constant nanogelexposure to cells in vitro.

Plasmacytoid dendritic cells are potent producers of IFN-α, aninflammatory cytokine that exacerbates autoimmunity in mouse lupusmodels and human lupus patients. pDCs that were briefly treated withnanoparticles for 30 minutes and then challenged with CpGoligonucleotides had marked reduction in IFN-α production (FIG. 19B).Collectively, these results indicate that pharmacologic agent deliverywith nanoparticles to conventional and plasmacytoid dendritic cells canattenuate their ability to promote aberrant inflammatory responses whichcontribute to autoimmunity.

Example 17: Nanolipogels Exhibit Improved Properties Over ConventionalPLGA Nanoparticles

Materials and Methods

The efficacy of nanolipogels was compared to PLGA using both in vivo andin vitro evaluation. For in vivo evaluation, NZB/W F1 lupus-prone micewere treated with 0.625 mg MPA per kg animal with nanolipogels or PLGAparticles. Survival time was monitored to determine and compare in vivoefficacy between particles.

For in vitro studies, bone marrow derived dendritic cells (BMDCs) werecultured. On day 1 of the culture, MPA loaded within nanolipogel or PLGAparticles were added to cells. On day 6 of the culture,lipopolysaccharide was added, and dendritic cell surface markers weremeasured by flow cytometry. Levels of cytokine production was measuredfrom the supernatant by ELISA. For nanoparticle internalization studies,BMDCs were incubated for 1 hr with nanolipogels or PLGA particlescontaining fluorescent rhodamine tracer. The extent of nanoparticleinternalization was assessed with flow cytometry, by quantifying thepercentage of CD11c⁺ cells that were positively labeled with rhodamine.

Results

The efficacy of MP A-loaded nanolipogel was also compared toconventional MPA-loaded PLGA nanoparticles and free MPA in the model forSLE.

The results show that nanolipogel loaded particles, but not PLGA loadedparticles or free drug increased survival time to treated mice (FIGS.20A and 20B).

The results also show that nanolipogel loaded particles induce a greaterreduction of immunostimulatory surface molecules on dendritic cells (%CD40 positive and % CD80 positive) than PLGA loaded particles after LPSchallenge (FIGS. 20C and 20D). Similar results were observed with CD86,MHC I, and MHC II.

The results also show that nanolipogel loaded particles induce a greaterreduction in proinflammatory cytokine production (i.e., IFN-γ, IL-12p70, and TNF-α) in dendritic cells compared to PLGA loaded particles andfree drug after LPS challenge (FIGS. 20E-20G).

It is believed that this result may be due in part to increased cellinternalization of nanolipogels compared to PLGA nanoparticles (FIG.20H). Together, this data shows that nanolipogels exhibit increasedefficacy over conventional PLGA nanoparticles.

CONCLUSION

Examples show development of a hydrogel-based nanoparticle(“nanolipogel”) loaded with the immunosuppressant MPA. The studies alsoshow that drug loaded nanogels can be used to achieve therapeuticimmunosuppression in a lupus-prone mouse model. The use of thesenanolipogels enhanced the potency of MPA therapy, as an equivalent doseof MPA administered without nanoparticles did not provide therapeuticbenefit. Lupus median survival was enhanced by approximately 3 months,with delay in development of nephritis and partial attenuation inautoantibody production. The administration of the drug in the nanogelsrequires significantly smaller dose for therapeutic efficacy, whencompared to administration of the free drug. Significantly higher doses(>16-fold) of free drug could not achieve the same therapeutic result assmaller amounts of drug administered within nanoparticles.

These nanoparticles have defined interactions with immune cell subsetsinvolved with lupus pathogenesis, and these immune cells can be affectedby nanoparticle therapy. The data shows the ability of nanoparticles tomore effectively traffic to spleen and target immune cell subsetsinvolved with lupus pathogenesis than free drug. This enhancement in MPAbioavailability likely contributes to the improved therapeutic benefitobserved with particles over freely administered MPA. All immune cellsubsets appear to associate with nanoparticles to some extent in vivo,which results in overall reductions in inflammatory CD4 T cell responsesat 36-40 weeks of age and ultimately delayed nephritis. Dendritic cellinternalization of nanoparticles may contribute to thisimmunosuppression.

One of ordinary skill in the art can readily extend the results seenwith nanogels in the examples, to delivery of pharmaceutical agent totreat and/or ameliorate the symptoms of other autoimmune diseases.

Modifications and variations of the compositions and methods ofmanufacture and use thereof will be obvious to those skilled in the artfrom the foregoing detailed description and are intended to come withinthe scope of the appended claims. All references are specificallyincorporated.

The invention claimed is:
 1. A nanolipogel comprising a polymeric matrixcore, and a lipid shell; wherein the nanolipogel is loaded with ananti-inflammatory cytokine and a growth factor each dispersed within thepolymeric matrix, dispersed in the lipid shell, and/or bound to thelipid shell.
 2. The nanolipogel of claim 1, wherein theanti-inflammatory cytokine is IL-2, interleukin (IL)-1 receptorantagonist, IL-4, IL-6, IL-10, IL-11, or IL-13.
 3. The nanolipogel ofclaim 1, wherein the growth factor is TNF, CSF, GM-CSF or G-CSF.
 4. Thenanolipogel of claim 1, wherein the nanolipogel comprises at least onetargeting moiety that increases the nanolipogel's specificity for atarget cell.
 5. The nanolipogel of claim 4, wherein the targeting moietyis specific for CD4-positive T cells.
 6. The nanolipogel of claim 5,wherein the targeting moiety is an anti-CD4 antibody or antigen bindingfragment thereof.
 7. The nanolipogel of claim 4, wherein the targetingmoiety is specific for antigen presenting cells selected frommacrophages, B cells, and monocytes.
 8. The nanolipogel of claim 7,wherein the antigen presenting cells are dendritic cells.
 9. Thenanolipogel of claim 1, wherein the polymeric matrix, the lipid shell,or both are crosslinked or wherein the polymeric matrix is formed ofnon-crosslinkable polymers.
 10. The nanolipogel of claim 1, wherein thepolymeric matrix comprises polymer selected from poly(lactic acid),poly(glycolic acid), poly(lactic acid-co-glycolic acids),polyhydroxyalkanoates; polycaprolactones; poly(orthoesters);polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones);poly(glycolide-co-caprolactones); polycarbonates; polyamides,polypeptides, and poly(amino acids); polyesteramides; poly(dioxanones);poly(alkylene alkylates); hydrophilic polyethers; polyurethanes;polyetheresters; polyacetals; polycyanoacrylates; polysiloxanes;poly(oxyethylene)/poly(oxypropylene) copolymers; polyketals;polyphosphates; polyhydroxyvalerates; polyalkylene oxalates;polyalkylene succinates; poly(maleic acids), polyvinyl alcohols,polyvinylpyrrolidone; poly(alkylene oxides); celluloses, polyacrylicacids, albumin, collagen, gelatin, prolamines, polysaccharides,derivatives, copolymers, and blends thereof.
 11. The nanolipogel ofclaim 1, wherein the nanolipogel further comprises a host moleculedispersed within or covalently bound to the polymeric matrix core,wherein the host molecule is selected from polysaccharides, cryptands,cryptophanes, cavitands, crown ethers, dendrimers, ion-exchange resins,calixarenes, valinomycins, nigericins, catenanes, polycatenanes,carcerands, cucurbiturils, spherands, carbon nanotubes, fullerenes, andgraphene-based host materials.
 12. The nanolipogel of claim 1, where thelipid shell comprises one or more concentric lipid layers, optionallycrosslinked, wherein the lipids can be neutral, anionic or cationiclipids at physiologic pH.
 13. The nanolipogel of claim 1, wherein thelipid shell comprises a lipid selected from cholesterol, phospholipids,lysolipids, lysophospholipids, and sphingolipids, and derivativesthereof.
 14. The nanolipogel of claim 13, comprising lipid selected fromphosphatidylcholine; phosphatidylserine, phosphatidylglycerol,phosphatidylinositol; glycolipids; sphingomyelin, ceramidegalactopyranoside, gangliosides, cerebrosides; fatty acids, sterols;1,2-diacyl-sn-glycero-3-phosphoethanolamines,1,2-dihexadecylphosphoethanolamine, 1,2-di stearoylphosphatidylcholine,1,2-dipalmitoylphosphatidylcholine, 1,2-dimyristoylphosphatidylcholine,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts,dimethyldioctadecyl ammonium bromide, 1,2-diacyloxy-3-trimethylammoniumpropanes, N-[1-(2,3-dioloyloxy)propyl]-N,N-dimethyl amine,1,2-diacyloxy-3-dimethylammonium propanes,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride,1,2-dialkyloxy-3-dimethylammonium propanes,dioctadecylamidoglycylspermine,3-[N—(N′,N′-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol);2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanaminiumtrifluoro-acetate (DOSPA), β-alanyl cholesterol, cetyltrimethylammoniumbromide (CTAB), diC₁₄-amidine,N-tert-butyl-N′-tetradecyl-3-tetradecylamino-propionamidine,N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG),ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride,1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), andN,N,N′,N′-tetramethyl-,N′-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butanediammonium iodide,1-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazoliniumchloride derivatives, and 2,3-dialkyloxypropyl quaternary ammoniumderivatives containing a hydroxyalkyl moiety on the quaternary amine,for example, 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide(DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DORIE), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropyl ammonium bromide(DOME-HP), 1,2-dioleyl-oxy-propyl-3-dimethyl-hydroxybutyl ammoniumbromide (DORIE-HB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentylammonium bromide (DORIE-Hpe),1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide(DMRIE), 1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammoniumbromide (DPRIE), and 1,2-disteryloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DSRIE).
 15. The nanolipogel of claim 1, wherein thelipid is a PEGylated derivative of a neutral, anionic, or cationiclipid.
 16. The nanolipogel of claim 1, wherein the nanolipogel displayspolyethylene glycol chains on its surface.
 17. The nanolipogel of claim1, wherein the lipid shell is unilamellar.
 18. The nanolipogel of claim10 wherein the polymeric matrix comprises polymer selected frompoly3-hydroxybutyrate and poly4-hydroxybutyrate.
 19. The nanolipogel ofclaim 11 wherein the host molecule is selected from amyloses,cyclodextrins, and compounds containing a plurality of aldose rings anddisaccharides.
 20. The nanolipogel of claim 14 comprising lipid selectedfrom1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazoliniumchloride (DOTIM) and1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazoliniumchloride (DPTIM).